Immunogenic complexes of polyanionic carbomers and env polypeptides and methods of manufacture and use thereof

ABSTRACT

The present invention relates to immunogenic complexes formed between polyanionic carbomers and Env polypeptides. Uses of the immunogenic complexes in applications including inducing an immune response and immunization generally are described. Methods of forming and manufacture of the immunogenic complexes are also described. The present invention also relates to immunogenic compositions including low viscosity, polyanionic carbomers and Env polypeptides. Uses of such immunogenic compositions in applications including inducing an immune response and immunization generally are described. Methods of manufacture of such immunogenic compositions are also described.

CROSS REFERENCE TO RELATED APPLICATION

This application claims the benefit of priority under 35 U.S.C. §119(e)to U.S. Provisional Application No. 61/559,512, filed Nov. 14, 2011. Thedisclosure of the above application is incorporated herein by referencein its entirety.

GOVERNMENT SUPPORT

This invention was made in part with U.S. Government support underHIVRAD grant 5P01 AI066287 awarded by the NIAID, NIH. The Government hascertain rights in the invention.

TECHNICAL FIELD

Immunogenic compositions comprising complexes between polyanioniccarbomers and Env polypeptides are described, as are uses of theseimmunogenic compositions and methods of forming and manufacturing suchcomplexes. Immunogenic compositions comprising low viscosity,polyanionic carbomers and Env polypeptides are described, as are uses ofthese immunogenic compositions and methods of forming and manufacturingsuch compositions.

BACKGROUND

Acquired immune deficiency syndrome (AIDS) is recognized as one of thegreatest health threats facing modern medicine. There is, as yet, nocure for this disease.

In 1983-1984, three groups independently identified the suspectedetiological agent of AIDS. See, e.g., Barre-Sinoussi et al. (1983)Science 220:868-871; Montagnier et al., in Human T-Cell Leukemia Viruses(Gallo, Essex & Gross, eds., 1984); Vilmer et al. (1984) The Lancet1:753; Popovic et al. (1984) Science 224:497-500; Levy et al. (1984)Science 225:840-842. These isolates were variously calledlymphadenopathy-associated virus (LAV), human T-cell lymphotropic virustype III (HTLV-III), or AIDS-associated retrovirus (ARV). All of theseisolates are strains of the same virus, and were later collectivelynamed Human Immunodeficiency Virus (HIV). With the isolation of arelated AIDS-causing virus, the strains originally called HIV are nowtermed HIV-1 and the related virus is called HIV-2. See, e.g., Guyaderet al. (1987) Nature 326:662-669; Brun-Vezinet et al. (1986) Science233:343-346; Clavel et al. (1986) Nature 324:691-695.

A great deal of information has been generated about the HIV virus;however, to date an effective vaccine has not been identified. Severaltargets for vaccine development have been examined including the Env andGag gene products encoded by HIV. Gag gene products include, but are notlimited to, Gag-polymerase and Gag-protease. Env gene products include,but are not limited to, monomeric gp120 polypeptides, oligomeric gp140polypeptides and gp160 polypeptides.

Use of HIV Env polypeptides in immunogenic compositions has beendescribed. (see, e.g., U.S. Pat. No. 5,846,546 to Hurwitz et al.,describing immunogenic compositions comprising a mixture of at leastfour different recombinant viruses that each expresses a different HIVenv variant; and U.S. Pat. No. 5,840,313 to Vahlne et al., describingpeptides which correspond to epitopes of the HIV-1 gp120 protein). Inaddition, U.S. Pat. No. 5,876,731 to Sia et al, describes candidatevaccines against HIV comprising an amino acid sequence of a T-cellepitope of Gag linked directly to an amino acid sequence of a B-cellepitope of the V3 loop protein of an HIV-1 isolate containing thesequence GPGR. However, none of these Env polypeptide base compositionshas been shown to provide a sufficient protective immune response to beuseful for an efficacious vaccine. Recently, G. Krashias et al.(Vaccine. 28:2482-2489, 2010) described a vaccine comprising gp140 andCARBOPOL™. G. Krashias et al. found that the CARBOPOL™ provided animproved immune response over alum as an adjuvant. However, G. Krashiaset al. found no detectable binding between gp140 and CARBOPOL™.

SUMMARY

The inventors have surprisingly found that, under appropriateconditions, polyanionic carbomers can form complexes with Envpolypeptides. The complexes show improved immunogenicity over existingadjuvanted HIV candidate vaccines.

Described herein are novel complexes between polyanionic carbomers andEnv polypeptides. One aspect of the disclosure includes immunogeniccompositions that comprise an Env polypeptide in complex with apolyanionic carbomer polymer. In one embodiment, the Env polypeptide isan HIV Env polypeptide or even an HIV-1 Env polypeptide. In anotherembodiment, which may be combined with the preceding embodiments, thepolyanionic carbomer polymer is free of benzene. In another embodiment,which may be combined with the preceding embodiments, the concentrationof the polyanionic carbomer polymer is between about 0.01% (w/v) andabout 2.0% (w/v), between about 0.01% (w/v) and about 0.5% (w/v), orbetween about 0.01% (w/v) and about 0.2% (w/v). In another embodiment,which may be combined with the preceding embodiments, the polyanionicpolymer comprises CARBOPOL 971P NF™, CARBOPOL 974P NF™, or combinationsthereof, or preferably CARBOPOL 971P NF™. In yet another embodiment,which may be combined with the preceding embodiments, the Envpolypeptide is trimeric. In certain embodiments which can be combinedwith the preceding embodiment, the Env polypeptide comprises one or moremutations. In certain embodiments which can be combined with thepreceding embodiments with one or more mutations, the one or moremutations are selected from mutations in the cleavage site that preventsthe cleavage of a gp140 polypeptide into a gp120 polypeptide and a gp41polypeptide, mutations in the glycosylation site, deletion of the V1region, deletion of the V2 region, and a combination of the foregoing.Preferably, the one or more mutations comprise a mutation in thecleavage site that prevents the cleavage of a gp140 polypeptide into agp120 polypeptide and a gp41 polypeptide and deletion of the V2 region.In certain embodiments which can be combined with the precedingembodiments, the Env polypeptide includes a gp160 Env polypeptide or apolypeptide derived from a gp160 Env polypeptide; a gp140 Envpolypeptide or a polypeptide derived from a gp140 Env polypeptide; or agp120 Env polypeptide or a polypeptide derived from a gp120 Envpolypeptide. In certain embodiments, the Env polypeptide comprises anamino acid sequence with at least 75% sequence identity, at least 80%sequence identity, at least 85% sequence identity, at least 90% sequenceidentity, at least 95% sequence identity, at least 97% sequenceidentity, at least 98% sequence identity, or at least 99% sequenceidentity to SEQ ID NOs: 22 or 23. In certain embodiments which can becombined with the preceding embodiments, the immunogenic compositionsfurther include a second Env polypeptide selected from a different HIVsubtype as the Env polypeptide. In certain embodiments which can becombined with the preceding embodiments which include a second Envpolypeptide, the second Env polypeptide and the Env polypeptide are inmixed complexes the polyanionic carbomer polymer. In certain embodimentswhich can be combined with the preceding embodiments which include asecond Env polypeptide, the second Env polypeptide is in a separatecomplex with a second polyanionic carbomer polymer or the polyanioniccarbomer polymer and the second polyanionic carbomer polymer are thesame type of polymer. In certain embodiments which can be combined withthe preceding embodiments which include a second Env polypeptide, theEnv polypeptide and the second Env polypeptide are derived from an HIVsubtype B strain and an HIV subtype C strain or vice-versa. In certainembodiments which can be combined with the preceding embodiments, theimmunogenic compositions further include one or more additional HIVpolypeptides. In certain embodiments which can be combined with thepreceding embodiments which include a one or more additional HIVpolypeptides, the one or more additional HIV polypeptides are selectedfrom the group comprising a Gag polypeptide, a Nef polypeptide, a Protpolypeptide, a Tat polypeptide, a Rev polypeptide, a Vif polypeptide, aVpr polypeptide, and a Vpu polypeptide. In certain embodiments which canbe combined with the preceding embodiments which include one or moreadditional HIV polypeptides, the one or more additional HIV polypeptidesinclude mutations that reduce or eliminate the activity of thepolypeptide without adversely affecting the ability of the additionalHIV polypeptides to generate an immune response. In certain embodimentswhich can be combined with the preceding embodiments, the immunogeniccomplexes further include an adjuvant. In certain embodiments which canbe combined with the preceding embodiments which include an adjuvant,the adjuvant is MF59.

Another aspect of the disclosure includes methods of generating theimmunogenic compositions above by (a) contacting the polyanioniccarbomer polymer with the Env polypeptide under conditions where the pHis below the pI of the Env polypeptide in a solution; (b) incubating thepolyanionic carbomer polymer with the Env polypeptide together to allowthe Env polypeptide to form a complex with the polyanionic carbomerpolymer. In one embodiment, the Env polypeptide is an HIV Envpolypeptide or even an HIV-1 Env polypeptide. In certain embodiments,which may be combined with the preceding embodiment, the pH is between 3and 6; between 3 and 5; or between 3 and 4. In another embodiment, whichmay be combined with the preceding embodiments, the polyanionic carbomerpolymer is free of benzene. In another embodiment, which may be combinedwith the preceding embodiment, the concentration of the polyanioniccarbomer polymer after contacting step (a) is between about 0.01% (w/v)and about 2.0% (w/v), between about 0.01% (w/v) and about 0.5% (w/v), orbetween about 0.01% (w/v) and about 0.2% (w/v). In another embodiment,which may be combined with the preceding embodiments, the polyanionicpolymer comprises CARBOPOL 971P NF™, CARBOPOL 974P NF™, or combinationsthereof, or preferably CARBOPOL 971P NF™. In yet another embodiment,which may be combined with the preceding embodiments, the Envpolypeptide is trimeric. In certain embodiments which can be combinedwith the preceding embodiments, the Env polypeptide comprises one ormore mutations. In certain embodiments which can be combined with thepreceding embodiments that include one or more mutations, the one ormore mutations are selected from mutations in the cleavage site thatprevents the cleavage of a gp140 polypeptide into a gp120 polypeptideand a gp41 polypeptide, mutations in the glycosylation site, deletion ofthe V1 region, deletion of the V2 region, and a combination of theforegoing. Preferably, the one or more mutations comprise a mutation inthe cleavage site that prevents the cleavage of a gp140 polypeptide intoa gp120 polypeptide and a gp41 polypeptide and deletion of the V2region. In certain embodiments which can be combined with the precedingembodiments, the Env polypeptide includes a gp160 Env polypeptide or apolypeptide derived from a gp160 Env polypeptide; a gp140 Envpolypeptide or a polypeptide derived from a gp140 Env polypeptide; or agp120 Env polypeptide or a polypeptide derived from a gp120 Envpolypeptide. In certain embodiments which can be combined with thepreceding embodiments, the Env polypeptide comprises an amino acidsequence with at least 75% sequence identity, at least 80% sequenceidentity, at least 85% sequence identity, at least 90% sequenceidentity, at least 95% sequence identity, at least 97% sequenceidentity, at least 98% sequence identity, or at least 99% sequenceidentity to SEQ ID NOs: 22 or 23. In certain embodiments which can becombined with the preceding embodiments, a second Env polypeptide isadded that is selected from a different HIV subtype as the Envpolypeptide to the solution. In certain embodiments which can becombined with the preceding embodiments that include a second Envpolypeptide, the second Env polypeptide is incubated with thepolyanionic carbomer polymer with the Env polypeptide to allow the Envpolypeptide and the second Env polypeptide to form complexes with thepolyanionic carbomer polymer simultaneously. In certain embodimentswhich can be combined with the preceding embodiments that include asecond Env polypeptide, the second Env polypeptide is in a separatecomplex with a second polyanionic carbomer polymer. In certainembodiments which can be combined with the preceding embodiments thatinclude a second polyanionic carbomer polymer, the polyanionic carbomerpolymer and the second polyanionic carbomer polymer are the same type ofpolymer. In certain embodiments which can be combined with the precedingembodiments that include a second Env polypeptide, the Env polypeptideand the second Env polypeptide are derived from an HIV subtype B strainand an HIV subtype C strain or vice-versa. In certain embodiments whichcan be combined with the preceding embodiments, the method furtherincludes a step of adding one or more additional HIV polypeptides. Incertain embodiments which can be combined with the preceding embodimentsthat include one or more additional HIV polypeptides, the one or moreadditional HIV polypeptides are selected from the group comprising a Gagpolypeptide, a Nef polypeptide, a Prot polypeptide, a Tat polypeptide, aRev polypeptide, a Vif polypeptide, a Vpr polypeptide, and a Vpupolypeptide. In certain embodiments which can be combined with thepreceding embodiments that include one or more additional HIVpolypeptides, the one or more additional HIV polypeptides includemutations that reduce or eliminate the activity of the polypeptidewithout adversely affecting the ability of the additional HIVpolypeptides to generate an immune response. In certain embodimentswhich can be combined with the preceding embodiments, the method furtherincludes a step of adding an adjuvant to the solution. In certainembodiments which can be combined with the preceding embodiments thatinclude an adjuvant, the adjuvant is MF59.

Yet another aspect of the disclosure includes methods of generating animmune response in a subject, comprising administering to said subjectan immunogenic composition according to the preceding composition aspector generated by the method according to the preceding method aspect,thereby generating the immune response to the Env polypeptide. In oneembodiment, the immunogenic composition is administered intramuscularly,intramucosally, intranasally, subcutaneously, intradermally,transdermally, intravaginally, intrarectally, orally or intravenously.In certain embodiments which can be combined with the precedingembodiment, the subject is a mammal. Preferably, the mammal is a human.In certain embodiments which can be combined with the precedingembodiments, the immune response includes a humoral immune response. Incertain embodiments which can be combined with the precedingembodiments, the immune response includes a cellular immune response.

Still another aspect of the disclosure includes methods of generating anenhanced immune response in a subject by (a) transfecting cells of saidsubject with a gene delivery vector for expression of an Envpolypeptide, under conditions that permit the expression of the Envpolypeptide, thereby generating an immune response to the Envpolypeptide; (b) administering to said subject an immunogeniccomposition according to the preceding composition aspect or generatedby the method according to the preceding method aspect, therebyenhancing the immune response to the Env polypeptide.

Another aspect of the disclosure includes methods of generating anenhanced immune response in a subject previously having had a genedelivery vector for expression of an Env polypeptide transfected intocells of the subject under conditions that permitted the expression ofthe Env polypeptide thereby having generated an immune response to theEnv polypeptide, comprising administering to said subject an immunogeniccomposition according to an immunogenic composition according to thepreceding composition aspect or generated by the method according to thepreceding method aspect, thereby enhancing the immune response to theEnv polypeptide. In one embodiment, the immunogenic composition isadministered intramuscularly, intramucosally, intranasally,subcutaneously, intradermally, transdermally, intravaginally,intrarectally, orally or intravenously. In certain embodiments which canbe combined with the preceding embodiment, the subject is a mammal.Preferably, the mammal is a human. In certain embodiments which can becombined with the preceding embodiments, the immune response includes ahumoral immune response. In certain embodiments which can be combinedwith the preceding embodiments, the immune response includes a cellularimmune response.

Described herein are novel compositions inclusing low viscosity,polyanionic carbomers and Env polypeptides. One aspect of the disclosureincludes immunogenic compositions that comprise an Env polypeptide witha low viscosity, polyanionic carbomer polymer. In one embodiment, theEnv polypeptide is an HIV Env polypeptide or even an HIV-1 Envpolypeptide. In another embodiment, which may be combined with thepreceding embodiments, the polyanionic carbomer polymer is free ofbenzene. In another embodiment, which may be combined with the precedingembodiments, the concentration of the polyanionic carbomer polymer isbetween about 0.01% (w/v) and about 2.0% (w/v), between about 0.01%(w/v) and about 0.5% (w/v), or between about 0.01% (w/v) and about 0.2%(w/v). In another embodiment, which may be combined with the precedingembodiments, the low viscosity, polyanionic polymer comprises apolyanionic polymer with an average viscosity of less than 25,000 cP(25° C., Brookfield RVT, 20 rpm, neutralized to pH 7.3-7.8, 0.5 wt %mucilage, spindle #6), less than 20,000 cP, less than less than 15,000cP, or the low viscosity, polyanionic polymer is CARBOPOL 971P NF™. Inyet another embodiment, which may be combined with the precedingembodiments, the Env polypeptide is trimeric. In certain embodimentswhich can be combined with the preceding embodiment, the Env polypeptidecomprises one or more mutations. In certain embodiments which can becombined with the preceding embodiments with one or more mutations, theone or more mutations are selected from mutations in the cleavage sitethat prevents the cleavage of a gp140 polypeptide into a gp120polypeptide and a gp41 polypeptide, mutations in the glycosylation site,deletion of the V1 region, deletion of the V2 region, and a combinationof the foregoing. Preferably, the one or more mutations comprise amutation in the cleavage site that prevents the cleavage of a gp140polypeptide into a gp120 polypeptide and a gp41 polypeptide and deletionof the V2 region. In certain embodiments which can be combined with thepreceding embodiments, the Env polypeptide includes a gp160 Envpolypeptide or a polypeptide derived from a gp160 Env polypeptide; agp140 Env polypeptide or a polypeptide derived from a gp140 Envpolypeptide; or a gp120 Env polypeptide or a polypeptide derived from agp120 Env polypeptide. In certain embodiments, the Env polypeptidecomprises an amino acid sequence with at least 75% sequence identity, atleast 80% sequence identity, at least 85% sequence identity, at least90% sequence identity, at least 95% sequence identity, at least 97%sequence identity, at least 98% sequence identity, or at least 99%sequence identity to SEQ ID NOs: 22 or 23. In certain embodiments whichcan be combined with the preceding embodiments, the immunogeniccompositions further include a second Env polypeptide selected from adifferent HIV subtype as the Env polypeptide. In certain embodimentswhich can be combined with the preceding embodiments which include asecond Env polypeptide, the Env polypeptide and the second Envpolypeptide are derived from an HIV subtype B strain and an HIV subtypeC strain or vice-versa. In certain embodiments which can be combinedwith the preceding embodiments, the immunogenic compositions furtherinclude one or more additional HIV polypeptides. In certain embodimentswhich can be combined with the preceding embodiments which include a oneor more additional HIV polypeptides, the one or more additional HIVpolypeptides are selected from the group comprising a Gag polypeptide, aNef polypeptide, a Prot polypeptide, a Tat polypeptide, a Revpolypeptide, a Vif polypeptide, a Vpr polypeptide, and a Vpupolypeptide. In certain embodiments which can be combined with thepreceding embodiments which include one or more additional HIVpolypeptides, the one or more additional HIV polypeptides includemutations that reduce or eliminate the activity of the polypeptidewithout adversely affecting the ability of the additional HIVpolypeptides to generate an immune response. In certain embodimentswhich can be combined with the preceding embodiments, the immunogeniccompositions further include an adjuvant. In certain embodiments whichcan be combined with the preceding embodiments which include anadjuvant, the adjuvant is MF59.

Yet another aspect of the disclosure includes methods of generating animmune response in a subject, comprising administering to said subjectan immunogenic composition according to the preceding compositionaspect, thereby generating the immune response to the Env polypeptide.In one embodiment, the immunogenic composition is administeredintramuscularly, intramucosally, intranasally, subcutaneously,intradermally, transdermally, intravaginally, intrarectally, orally orintravenously. In certain embodiments which can be combined with thepreceding embodiment, the subject is a mammal. Preferably, the mammal isa human. In certain embodiments which can be combined with the precedingembodiments, the immune response includes a humoral immune response. Incertain embodiments which can be combined with the precedingembodiments, the immune response includes a cellular immune response.

Still another aspect of the disclosure includes methods of generating anenhanced immune response in a subject by (a) transfecting cells of saidsubject with a gene delivery vector for expression of an Envpolypeptide, under conditions that permit the expression of the Envpolypeptide, thereby generating an immune response to the Envpolypeptide; (b) administering to said subject an immunogeniccomposition according to the preceding composition aspect, therebyenhancing the immune response to the Env polypeptide.

Another aspect of the disclosure includes methods of generating anenhanced immune response in a subject previously having had a genedelivery vector for expression of an Env polypeptide transfected intocells of the subject under conditions that permitted the expression ofthe Env polypeptide thereby having generated an immune response to theEnv polypeptide, comprising administering to said subject an immunogeniccomposition according to an immunogenic composition according to thepreceding composition aspect, thereby enhancing the immune response tothe Env polypeptide. In one embodiment, the immunogenic composition isadministered intramuscularly, intramucosally, intranasally,subcutaneously, intradermally, transdermally, intravaginally,intrarectally, orally or intravenously. In certain embodiments which canbe combined with the preceding embodiment, the subject is a mammal.Preferably, the mammal is a human. In certain embodiments which can becombined with the preceding embodiments, the immune response includes ahumoral immune response. In certain embodiments which can be combinedwith the preceding embodiments, the immune response includes a cellularimmune response.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows immunoblots of the Env polypeptide-CARBOPOL™ complexesafter incubation for various periods of time. Panel (A) shows from leftto right (1) molecular weight markers, (2) Env polypeptide complex—0hours, (3) Env polypeptide complex—1 hour at 4° C., (4) Env polypeptidecomplex—2 hours at 4° C., (5) Env polypeptide complex—3 hours at 4° C.,and (6) Env polypeptide complex—4 hours at 4° C. Panel (B) shows fromleft to right (1) molecular weight markers, (2) Env polypeptidecomplex—1 hour at 4° C., (3) Env polypeptide complex—1 hour at 20° C.,(4) Env polypeptide in PBS—1 hour at room temperature (25° C.), (5)molecular weight markers, (6) Env polypeptide complex—1 hour at 30° C.,(7) Env polypeptide complex—1 hour at 37° C., and (8) Env polypeptide inPBS—1 hour at 37° C. The final concentration of CARBOPOL™ in the gelswas ˜0.1%.

FIG. 2 shows the dynamic light scattering analysis (averaging tenmeasurements) for the CARBOPOL™ alone (left) or CARBOPOL™ in complexwith the Env (gp140) polypeptide. The CARBOPOL™ alone displayed ahydrodynamic radius of ˜68 nm. The CARBOPOL™+gp140 showed a radius of˜86 nm, indicating that the CARBOPOL™ and the gp140 were interacting ina higher order complex. The final concentration of CARBOPOL™ in the gelswas ˜0.1%.

FIG. 3 shows ELISA assays testing binding of Env polypeptide incubatedwith 0.5% CARBOPOL™, 1:1 (v/v), (final conc. of CARBOPOL™ 0.25%) for onehour (dark grey open boxes) or three hours (light grey open boxes) atroom temperature (25° C.). Env polypeptide without CARBOPOL™ (ascontrol) was similarly incubated (closed boxes). The y-axis shows OD 450nm and the x-axis shows concentration of gp120 (g/ml). (A) shows thegp120 samples binding to CD4-IgG2 (surrogate for receptor CD4). (B)shows the gp120 samples binding to b12 (a CD4-binding site neutralizingmonoclonal antibody, mAb). (C) shows binding to 17b mAb (a CD4-inducedmonoclonal antibody), in presence or absence of soluble CD4, sCD4. TheCD4gG2 & b12 mAb binding shows that the conformational receptor bindingsite was antigenically intact, despite incubation with CARBOPOL™. The17b mAb binding confirmed that the protein was able to undergoCD4-induced conformation change, a key aspect of functional Envpolypeptide.

FIG. 4 shows a chart of the response units (RU—y-axis) measured viasurface plasmon resonance for the Env polypeptide alone or in complexwith polyanionic carbomers (CARBOPOL™). Binding to soluble CD4 (sCD4),bound to the sensor chip, is shown on the left with white bars. Bindingto a glycan-dependent, monoclonal antibody (2G12), bound to the sensorchip, is shown on the right with shaded gray bars.

FIG. 5 shows a chart of the antibodies in sera as measured withgp120-binding ELISA (geometric mean titer—y axis) from a rabbit studycomparing Env polypeptide (SF162 gp140) adjuvanted with CARBOPOL™ orMF59™ or CARBOPOL™+MF59™. The geometric mean titer at two-weekspost-second immunization (2wp2) is shown with white bars. The geometricmean titer at two-weeks post-third immunization (2wp3) is shown withlight gray bars. The geometric mean titer at two-weeks post-fourthimmunization (2wp4) is shown with black bars.

FIG. 6 shows a chart of the avidity of the gp120-specific serumantibodies assessed using ammonium thiocyanate ELISA (avidity index—yaxis). The sera are from a rabbit study comparing Env polypeptide (SF162gp140) adjuvanted with CARBOPOL™ or MF59™ or CARBOPOL™+MF59™. Theavidity index at two-weeks post-second immunization (2wp2) is shown withwhite bars. The avidity index at two-weeks post-third immunization(2wp3) is shown with light gray bars. The avidity index at two-weekspost-fourth immunization (2wp4) is shown with black bars.

FIGS. 7A-B show results for the neutralization potential of Env-specificantibodies produced from the immunization regimens in a rabbit studycomparing Env polypeptide (SF162 gp140) adjuvanted with CARBOPOL™ orMF59™ or CARBOPOL™+MF59™. Each graph shows ID50 titers of antibodiesfrom post-third (p3) and post-fourth (p4) immunization for (a)immunization with Env polypeptides with MF59™, (b) immunization with Envpolypeptides-polyanionic carbomer complexes, and (c) immunization withEnv polypeptides-polyanionic carbomer complexes with MF59™. FIG. 7Ashows the neutralization potentials against Tier 1a and Tier 1bisolates. FIG. 7B shows the neutralization potentials against Tier 2isolates and the control.

FIGS. 8A-B show a heat map that shows the breadth and potency (ID50titers) of serum neutralization of HIV-1 pseudoviruses. The results fromeach of the five rabbits in each group are shown. Samples in blackdemonstrated 50% neutralization with a serum dilution from 1,000 to9,999; samples in dark grey demonstrated 50% neutralization with a serumdilution from 100 to 999; and samples shaded in light grey demonstrated50% neutralization with a serum dilution from 20 to 99. FIG. 8A showsthe breadth and potency against Tier 1a and Tier 1b isolates. FIG. 8Bshows the breadth and potency against Tier 2 isolates.

FIGS. 9A-E show neutralization ID50 titers of against various isolates.FIG. 9A shows the neutralization ID50 titers of against two Tier 1aisolates (2wp3 (p3), 2wp4 (p4), & 2wp5 (p5)): MW965.26 (a subtype C) andSF162.LS (a subtype B). FIG. 9B shows the neutralization ID50 titers ofagainst another Tier 1a isolate (2wp3 (p3), 2wp4 (p4), & 2wp5 (p5)): MN(a subtype B). FIG. 9C shows the neutralization ID50 titers of againsttwo Tier 1b isolates (2wp3 (p3), 2wp4 (p4), & 2wp5 (p5)): Bal.26 (asubtype C) and TV1.21 (a subtype B). FIG. 9D shows the neutralizationID50 titers of against two Tier 2 isolates (2wp3 (p3), 2wp4 (p⁴), & 2wp5(p5)): ZM249M.PL1 (a subtype C) and Du156.12 (a subtype C). FIG. 9Eshows the neutralization ID50 titers of against another Tier 2 isolate(2wp3 (p3), 2wp4 (p4), & 2wp5 (p5)): Du422.1 (a subtype C).

FIG. 10 shows total antibody-binding titers against TV1 gp140 Envpolypeptide as measured by gp120-binding ELISA. The background titer forthe prebleeds (as control) is also included. The antibody titers weredetermined by ELISA using TV1 gp140 Env polypeptide as the coatingprotein. The data values shown represent geometric mean titers (GMT) offive rabbits individually assayed in triplicates per group.

FIG. 11 shows antibody avidity of sera collected from all ten groups ofrabbits from Example 6 that were immunized with Env polypeptide, eithermonovalent or multivalent, adjuvanted with MF59™ (and in one case withMF59™ and CARBOPOL™). The groups are from left to right: Du422.1 gp140,Du156.12 gp140, CAP45 gp140, ZM249M.PL1 gp140, HIV-25711-2 (EF117272)gp140, CAP255 (EF203982) gp140, CAP239 (EF203983) gp140, ZM249M.PL1gp140+CAP239 (EF203983) gp140+Du422.1 gp140 (with MF59™ only),ZM249M.PL1 gp140+CAP239(EF203983) gp140+Du422.1 gp140 (with MF59™ andCARBOPOL™), and TV1 gp140. The avidity for each group is shown (fromleft to right) in increasing shades of grey for prebleed, two-weeks postsecond immunization (2wp2), two-weeks post third immunization (2wp3),two-weeks post fourth immunization (2wp4), and two-weeks post fifthimmunization (2wp5). Avidity was determined by NH₄SCN displacement ELISAusing TV1 gp140 Env polypeptide as the coating antigen as described byI. K. Srivastava et al. (J. Virol. 2002).

FIG. 12A-B shows a heat map that shows the breadth and potency (ID50titers) of serum neutralization of HIV-1 pseudoviruses using serumcollected at two-weeks post third immunization. Samples in blackdemonstrated 50% neutralization with a serum dilution of greater than10,000; samples in dark grey with white colored numbers demonstrated 50%neutralization with a serum dilution of 1,000 to 9,999; samples in darkgrey demonstrated 50% neutralization with a serum dilution from 100 to999; and samples shaded in light grey demonstrated 50% neutralizationwith a serum dilution from 20 to 99.

FIG. 13A-C shows a heat map that shows the breadth and potency (ID50titers) of serum neutralization of HIV-1 pseudoviruses using serumcollected at two-weeks post fourth immunization for an extended panel ofstrains. Samples in black demonstrated 50% neutralization with a serumdilution from 1,000 to 9,999; samples in dark grey demonstrated 50%neutralization with a serum dilution from 100 to 999; and samples shadedin light grey demonstrated 50% neutralization with a serum dilution from20 to 99.

FIGS. 14A-E shows ID50 neutralization titers of sera obtained two weekspost third immunization (p3) and two weeks post fourth (p4)immunization, as determined using a HIV-1 pseudovirus assay. FIG. 14Ashows neutralization titers against two Tier 1A isolates: MW965.26(subtype C) (left) and SF162.LS (subtype B) (right). FIG. 14B showsneutralization titers against a third Tier 1A isolate: MN.2 (subtype B).FIG. 14C shows neutralization titers against two Tier 1B isolates:Bal.26 (subtype B) (left) and TV1.21 (subtype C) (right). FIG. 14D showsneutralization titers against two Tier 2 isolates: ZM249M.PL1 (subtypeC) (left) and Du165.12 (subtype C) (right). FIG. 14E showsneutralization titers against a third Tier 2 isolate: Du422.1 (subtypeC).

FIG. 15 shows binding-antibody titers for rabbit sera collected from allnine groups measured with gp120-binding ELISA (geometric mean titer—yaxis). The groups are from left to right: The groups are from left toright: Du156.12 gp140, Du422.1 gp140, ZM249M.PL1 gp140, CAP239 gp140,TV1 gp140, TV1 gp140ΔV2, SF162 gp140ΔV2, ZM249M.PL1 gp140+CAP239gp140+Du422.1 gp140+TV1 gp140 (group 8, multivalent administration), andCAP239 gp140/Du422.1 gp140/ZM249M.PL1 gp140/TV1 gp140 (group 9,sequential administration). The geometric mean titer for each group isshown (from left to right) in increasing shades of grey for prebleed,two-weeks post second immunization (2wp2), two-weeks post thirdimmunization (2wp3), two-weeks post fourth immunization (2wp4), andbleed out.

FIG. 16 shows antibody avidity of rabbit sera collected from all ninegroups. The groups are from left to right: Du156.12 gp140, Du422.1gp140, ZM249M.PL1 gp140, CAP239 gp140, TV1 gp140, TV1 gp140ΔV2, SF162gp140ΔV2, ZM249M.PL1+CAP239+Du422.1+TV1 gp140 (group 8), and CAP239gp140/Du422.1 gp140/ZM249M.PL1 gp140/TV1 gp140 (group 9). The avidityindex for each group is shown (from left to right): two-weeks postsecond immunization (2wp2), two-weeks post third immunization (2wp3),two-weeks post fourth immunization (2wp4), and bleed out. Avidity wasdetermined by NH4SCN displacement ELISA using TV1 gp140 Env polypeptideas the coating antigen as described by I. K. Srivastava et al. (J.Virol. 2002).

FIGS. 17A-F show neutralization potential (ID50 titers, y-axis) of theantibodies induced by immunization with each of the ten gp120 Envpolypeptide and the sequential immunization experiment against a panelof virus isolates (x-axis). FIG. 17A shows the neutralization potential(in ID50 titers) of Du156.12 gp120 (left) and Du422.1 gp120 (right).FIG. 17B shows the neutralization potential (in ID50 titers) ofZM249M.PL1 gp120 (left) and CAP45 (EF203960) gp120 (right). FIG. 17Cshows the neutralization potential (in ID50 titers) of CAP84 (EF203963)gp120 (left) and CAP239 (EF203983) gp120 (right). FIG. 17D shows theneutralization potential (in ID50 titers) of TV1 gp120 (left) and SF162gp120 (right). FIG. 17E shows the neutralization potential (in ID50titers) of TV1 gp140 (left) and SF162 gp140 (right). FIG. 17F shows theneutralization potential (in ID50 titers) of the sequential immunizationwith CAP239 gp120, Du422.1 gp120, ZM249.PL1 gp120, and TV1 gp120s (left)and all of the groups tested on a single chart (right).

FIG. 18A-B show a heat map that shows the breadth and potency (ID50titers) of serum neutralization of HIV-1 pseudoviruses using serumcollected at two-weeks post third immunization. Samples in dark greydemonstrated 50% neutralization with a serum dilution >10000; andsamples shaded in light grey demonstrated 50% neutralization with aserum dilution from 1000 to 9,999.

FIG. 19 shows results of monoclonal antibodies (mAbs) competition ELISAconducted against immobilized TV1gp140 Env polypeptide with pooledrabbit sera (1:100 dilution) collected 2 weeks post fourth immunizationwith subtype C gp120 (week 22), in order to map epitope specificities ofanti-Env antibodies elicited upon immunization. The isolates of Envpolypeptides tested are shown at the top. The epitope and mAbs used inthe competition assay are shown along the left.

FIGS. 20A-F show neutralization potential (ID50 titers, y-axis) of theantibodies induced by immunization of guinea pigs with each of the tenconstructs against a panel of virus isolates (x-axis). FIG. 20A showsthe neutralization potential (in ID50 titers) of Du156.12 gp120 (left)and Du422.1 gp120 (right). FIG. 20B shows the neutralization potential(in ID50 titers) of ZM249M.PL1 gp120 (left) and CAP45 (EF203960) gp120(right). FIG. 20C shows the neutralization potential of CAP84 (EF203963)gp120 (left) and the CAP239 (EF203983) gp120 (right). FIG. 20D shows theneutralization potential (in ID50 titers) of TV1 gp120 (left) and SF162gp120 (right). FIG. 20E shows the neutralization potential (in ID50titers) of TV1 gp140 (left) and SF162 gp140 (right). FIG. 20D shows theneutralization potential of all of the groups tested on a single chart.

FIG. 21 shows a heat map that shows the breadth and potency (in ID50titers) of serum neutralization of HIV-1 pseudoviruses using serumcollected at two-weeks post third immunization. Samples in dark greydemonstrated 50% neutralization with a serum dilution >10000; andsamples shaded in light grey demonstrated 50% neutralization with aserum dilution from 1000 to 9,999.

FIG. 22 shows results of monoclonal antibodies (mAbs) competition ELISAconducted against immobilized TV1 gp140 Env polypeptide with pooled sera(1:500 dilution) collected 2 weeks post third immunization (week 14)from the guinea pig study of Example 10, in order to map epitopespecificities of anti-Env antibodies elicited upon immunization. Theisolates of Env polypeptides tested are shown at the top. The epitopeand mAbs used in the competition assay are shown along the left.

FIG. 23 shows results of monoclonal antibodies (mAbs) competition ELISAconducted against immobilized TV1 gp140 Env polypeptide with pooled sera(1:500 dilution) collected 2 weeks post fourth immunization (week 26)from the guinea pig study of Example 10, in order to map epitopespecificities of anti-Env antibodies elicited upon immunization. Theisolates of Env polypeptides tested are shown at the top. The epitopeand mAbs used in the competition assay are shown along the left.

FIGS. 24A-K show the body weights (y axis) of fifty five rabbitsimmunized with gp120 Env polypeptide, adjuvanted with MF59™+CARBOPOL971™, at various time points after the immunization began (y axis). FIG.24A shows rabbits 1-5. FIG. 24B shows rabbits 6-10. FIG. 24C showsrabbits 11-15. FIG. 24D shows rabbits 16-20. FIG. 24E shows rabbits21-25. FIG. 24F shows rabbits 26-30. FIG. 24G shows rabbits 31-35. FIG.24H shows rabbits 36-40. FIG. 24I shows rabbits 41-45. FIG. 24J showsrabbits 46-50. FIG. 24K shows rabbits 51-55.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

HIV-1 envelope glycoprotein (Env) is the major viral protein exposed tohumoral immune response so it is an important target for vaccinedevelopment. Eliciting potent anti-HIV-1 neutralizing antibodies usingEnv has been complicated by various factors. A key factor is theantigenic variation and structural complexity of Env. Recombinant Envglycoproteins have shown sub-optimal immunogenicity in the absence of anadjuvant. Therefore, in addition to optimizing the design ofEnv-immunogens, identification of novel adjuvants and/or deliverysystems is important in generating vaccine-mediated protective immuneresponse against HIV.

Since Env is particularly labile and has conformation-dependentneutralization epitopes, adjuvants that do not denature or adverselymodify the antigenic structure are preferable. The following examplesdemonstrate that cross-linked, polyacrylic acid polymers (polyanioniccarbomers or CARBOPOL™) elicit a robust immune response when used incomplex with Env polypeptides. Polyacrylic acid polymers are especiallyadvantageous in that they can be combined with other adjuvants such asMF59™ to even further improve the immune response. Importantly, theexamples show an improvement in overall breadth and potency ofneutralizing antibodies when using polyanionic carbomers along withMF59™. Overall, the examples confirm that polyanionic carbomers can formcomplexes with Env without altering the antigenic structure or stabilityof the polypeptide and that the complexes elicit better immune responseupon vaccination alone or in combination with other adjuvants such asMF59™. While not limiting to theory, the improved immune response couldbe due to the polyanionic carbomers directing or presenting the Envpolypeptide to specific cells in the immune system and/or thepolyanionic carbomers stabilizing the Env polypeptides during storageand after vaccination. In addition, the Env polypeptide can beadjuvanted with low viscosity, polyanionic polymers with an averageviscosity of less than 25,000 cP (25° C., Brookfield RVT, 20 rpm,neutralized to pH 7.3-7.8, 0.5 wt % mucilage, spindle #6), less than20,000 cP, less than less than 15,000 cP. A preferred example of suchlow viscosity, polyanionic polymers is CARBOPOL 971P NF™.

The practice of the disclosed compositions and methods will onlyrequire, unless otherwise indicated, conventional methods of chemistry,biochemistry, molecular biology, immunology and pharmacology, within theskill of the art. Such techniques are explained fully in the literature.See, e.g., Remington's Pharmaceutical Sciences, 18th Edition (Easton,Pa.: Mack Publishing Company, 1990); Methods In Enzymology (S. Colowickand N. Kaplan, eds., Academic Press, Inc.); and Handbook of ExperimentalImmunology, Vols. I-IV (D. M. Weir and C. C. Blackwell, eds., 1986,Blackwell Scientific Publications); Sambrook, et al., Molecular Cloning:A Laboratory Manual (2nd Edition, 1989); Short Protocols in MolecularBiology, 4th ed. (Ausubel et al. eds., 1999, John Wiley & Sons);Molecular Biology Techniques: An Intensive Laboratory Course, (Ream etal., eds., 1998, Academic Press); PCR (Introduction to BiotechniquesSeries), 2nd ed. (Newton & Graham eds., 1997, Springer Verlag).

Polyanionic Carbomer Polymers

The polyanionic carbomer polymers to be used in the compositions andmethods disclosed herein are acrylic acid polymers. These acrylic acidpolymers may be homopolymers or copolymers. Polyanionic carbomerpolymers are commercially available under the trade name CARBOPOL™.Acrylic acid polymers are described, for example, in U.S. Pat. Nos.2,909,462 and 3,790,665.

While there are many polyanionic carbomer polymers to choose from whichwill form the complexes that have improved immunogenicity, the preferredpolyanionic carbomer polymers are those with lesser crosslinking andwhich are not formed in the presence of benzene so as to avoid residualbenzene (a potentially more toxic organic compound). Based upon thepreferred characteristics, CARBOPOL 971P NF™ polymer was selected as ithad residual ethyl acetate solvent (a class III solvent according to ICHguidelines) rather than benzene. We also considered 974P NF, which ischemically similar and has more toxicology and other supportive datashowing that it is safe, but since it is a cross linked polyacrylic acidof very high molecular weight, we decided to choose 971P NF since it wasa comparatively lightly cross-linked polymer and could aid solvation.Since regulatory and toxicological information are available for 974PNF, and they are likely applicable to 971P NF, we were satisfied tochoose the later in our studies.

The molecular weight range of these polymers depending upon the polymeris estimated to be from 740,000 to 4-5 million Daltons. There are nomethods available to measure the actual molecular weight of across-linked (i.e., 3-dimensional) polymer of this type, so the sizemust be estimated by other means such as dynamic light scattering, etc.The backbone of the homopolymer is the same, i.e., polymerized acrylicacid. The main differences relate to cross-link density and estimatedmolecular weight, rather than the cross-linker used. With very minoradjustments in the cross-linker density, one can produce a large numberof polyanionic carbomer polymers similar in gross molecular structurebut varying in application properties, for example, viscosity.Cross-link density can be varied by minor shifts in position of thecross-linker on the acrylic backbone. Because the actual cross-linkeritself has little, if any, effect on the biological properties of aparticular acrylic acid polymer, the Cosmetic, Toiletries and FragranceAssociation (CTFA) has adapted a family monograph, “carbomer,” for thesepolymers.

Polyanionic carbomer polymers, such as CARBOPOL™, PEMULEN™ and NOVEON™,are polymers within the scope of the invention. These particularpolyanionic carbomer polymers are cross-linked with polyalkenyl ethersor divinyl glycol.

Polyanionic carbomer polymers are flocculated powders of particlesaveraging about 0.2 micron in diameter. Each particle can be viewed as anetwork structure of polymer chains interconnected by crosslinks.Without the crosslinks, the primary particle would be a collection oflinear polymer chains, intertwined but not chemically bonded. Theselinear polymers are soluble in a polar solvent, such as water. In water,polyanionic carbomer polymers swell up to 1000 times their originalvolume (and ten times their original diameter) to form a gel whenexposed to a pH environment above 4-6. Since the pKa of these polymersis 6±0.5, the carboxylate groups on the polymer backbone ionize,resulting in repulsion between the negative particles, which adds to theswelling of the polymer. Cross-linked polymers do not dissolve in water.

Characteristics of Specific Types of Polyanionic Carbomer Polymers:

CARBOPOL 934 P™ is cross-linked with allyl sucrose and is polymerized insolvent benzene. CARBOPOL 971 P™ (71G, 974 P) are cross-linked withallyl penta erythritol and polymerized in ethyl acetate. Polycarbophilis cross-linked polymer in divinyl glycol and polymerized in solventbenzene. All the polymers fabricated in ethyl acetate are neutralized by1-3% potassium hydroxide. Though CARBOPOL 971 P™ and CARBOPOL 974 P™ aremanufactured by same process under similar conditions; the difference inthem is that CARBOPOL 971 P™ has slightly lower level of cross-linkingagent than CARBOPOL 974 P™. CARBOPOL 71 G™ is the granular form ofCARBOPOL™.

While the relationships between structure and properties have been ofinterest both academically and in industry. Different grades ofpolyanionic carbomer polymers exhibit different rheological properties,a reflection of the particle size, molecular weight between crosslinks(Mc), distributions of the Mc, and the fraction of the total units,which occur as terminal, i.e. free chain ends. The molecular weightsbetween adjacent crosslinks (Mc) are approximately inverselyproportional to the crosslinker density. These may be calculated fromthe functionality of the crosslinking monomer, the relative ratio ofacrylic acid to crosslinking monomer, and the efficiency of thecrosslinking reaction, assuming negligible chain ends. Alternatively,the molecular weight can be qualitatively compared to the rheologicalproperties of a swollen gel and/or from the equilibrium-swelling ratio.In simple terms, low viscosity, low rigidity polymer, such as CARBOPOL971 P™, have a higher Mc. Conversely, they have lower crosslinkerdensities. The higher the crosslinker level, the lower the equilibriumswelling ratio. In the network theory of elasticity, the elasticmodulus, G, is inversely proportional to the molecular weight betweencrosslinks (Mc).

CARBOPOL 971 P™ provides very low viscosities and excellent yield valuesat low usage levels. Suspensions formed with CARBOPOL 971 P™ will havelonger rheology. CARBOPOL 71 G™ polymers will give same viscosities andrheology as CARBOPOL 971 P™, but it is easier to handle and disperse dueto its granular nature.

Toxicity Details:

The polyanionic carbomer polymers, like other high molecular weightpolymers, demonstrate a low toxic and irritation potential based ontheir physical and chemical properties. Accordingly, such cross-linked,high molecular weight acrylic acid polymers have been found safe for usein a wide variety of cosmetics, detergents and pharmaceuticals byappropriate regulatory and nonregulatory bodies concerned with suchproducts.

Carbomer is the generic (i.e., nonproprietary) name adopted by USP-NF,United States Adopted Names Council (USAN) and CTFA for variousCARBOPOL™ polymers. The Cosmetic Ingredient Review (CIR) Expert Panel intheir assessment of the safety of the carbomers for cosmetic ingredientssummarized the toxicity of the carbomers as follows: Acute oral studieswith rats, guinea pigs, mice, and dogs showed that carbomers 910, -934,-940 and -941 have low toxicities when ingested. The inhalation LC₅₀ ofcarbomer 910 in albino rats was 1.71 mg/l. The dermal LC₅₀ of ratsexposed to carbomer 910 was greater than 3.0 g/kg. No mortalitiesoccurred in rabbits injected intravenously with 1%, 2% or 3% carbomer934 in aqueous solution at a dose of 5 ml/kg. Rabbits showed minimalskin irritation when tested with 100% carbomer 910 or -934, and zero tomoderate eye irritation when tested with carbomers 910, -934, -934P,-940, -941, and/or their various salts at concentrations of 0.20-100%.Subchronic feeding of rats with doses up to 5.0 g/kg/day carbomer 934(49 days) and of rats and dogs with up to 5.0% carbomer 934P in the diet(21 and/or 90 days) resulted in lower than normal body weights. In ratsfed carbomer 934P at dietary levels of 5.0% for 90 days, absolute liverweights and liver to body and brain weight ratios were reduced, but nopathological changes were observed. One of skill in the art can readilytake such issues into account when selecting which polyanionic carbomerpolymer to use in the compositions and methods disclosed herein.

Clinical studies with carbomer 934 (CARBOPOL 934 ™) and its varioussalts showed that these polymers have low potential for skin irritationand sensitization at concentrations of 0.5%, 5.0%, 10.0%, and 100%. Whentested on humans at 1.0% concentration, carbomers 940, -941, and theirvarious salts also demonstrated low potential for skin irritation andsensitization. Further, formulations containing up to 0.25% carbomer 934demonstrated low potential for human skin irritation, sensitization,phototoxicity, and photo-contact allergenicity. Clinical data forassessing the skin irritation and sensitization potential of carbomer940 and -941 were limited to studies in which concentrations of only1.0% were tested. Clinical data for assessing phototoxicity andphoto-contact allergenicity were limited to formulation studies in whichconcentrations of only 0.25% carbomer 934 were tested.

The CIR Expert Panel called attention to the presence of benzene as animpurity in the carbomers and recommended efforts to reduce it to thelowest possible level. In pursuit of this goal, Lubrizol AdvancedMaterials, Inc. has developed new CARBOPOL™ polymers which use alternatepolymerization solvent systems (e.g. ethyl acetate, cyclohexane, etc.).Thus, it is preferred to use polyanionic carbomer polymers such asCARBOPOL 971P NF™ that were not formed in the presence of benzene. Thesepolyanionic carbomer polymers are chemically identical to the benzenepolymerized polyanionic carbomer polymers and are therefore listed onthe U.S. Environmental Protection Agency's TSCA inventory as acrylicacid polymers or acrylic acid copolymers.

Preliminary toxicity test results on the ethyl acetate polymerizedpolymers are essentially similar to the previous products. They are notprimary irritants or sensitizers in human repeated patch tests. Thedermal LD50 was greater than 2000 mg/kg of body weight in the rabbit.Likewise it was minimally irritating to rabbit eyes. An acute oral LD50could not be obtained since intubation of enough polymer was notpossible. Results on a polyanionic carbomer polymers made in ethylacetate were consistent with the results expected for these polymers.That is, it was not an irritant to rabbit skin; undiluted polymer was amild to moderate irritant to the rabbit eyes, while a 1% solution(neutralized and unneutralized) were not eye irritants; application tohuman skin did not cause any skin irritation or sensitization. The LD50in rats is greater than 5,000 mg/kg and the dermal LD50 in rabbits isgreater than 2,000 mg/kg.

Given the similarity in the physical properties and structure ofpolyanionic carbomer polymers, one of skill in the art would recognizethat any polyanionic carbomer polymer will produce similar results asCARBOPOL 971P NF™. Therefore, one of skill in the art could readilyselect from any number of available polyanionic carbomer polymers toproduce the surprising result obtained herein based upon the foregoing.

When selecting a low viscosity, polyanionic polymer the averageviscosity will be less than 25,000 cP (25° C., Brookfield RVT, 20 rpm,neutralized to pH 7.3-7.8, 0.5 wt % mucilage, spindle #6), less than20,000 cP, or less than less than 15,000 cP. A preferred example of alow viscosity, polyanionic polymer is CARBOPOL 971P NF™.

Env Polypeptides

Env polypeptides include molecules derived from an envelope protein,preferably from HIV Env. The envelope protein of HIV-1 is a glycoproteinof about 160 kDa (gp160). During virus infection of the host cell, gp160is cleaved by host cell proteases to form gp120 and the integralmembrane protein, gp41. The gp41 portion is anchored in (and spans) themembrane bilayer of virion, while the gp120 segment protrudes into thesurrounding environment. As there is no covalent attachment betweengp120 and gp41, free gp120 is released from the surface of virions andinfected cells. Env polypeptides may also include gp140 polypeptides.Env polypeptides can exist as monomers or trimers.

Env polypeptides include molecules derived from the gp120 region of theEnv polypeptide. The primary amino acid sequence of gp120 isapproximately 511 amino acids, with a polypeptide core of about 60,000Daltons. The polypeptide is extensively modified by N-linkedglycosylation to increase the apparent molecular weight of the moleculeto 120,000 Daltons. The amino acid sequence of gp120 (and thereforegp140 and gp160) contains five relatively conserved domains interspersedwith five hypervariable domains. The positions of the 18 cysteineresidues in the gp120 primary sequence of the HIV-1_(HXB-2) strain, andthe positions of 13 of the approximately 24 N-linked glycosylation sitesin the gp120 sequence are common to most, if not all, gp120 sequences.The hypervariable domains contain extensive amino acid substitutions,insertions and deletions. Despite this variation, most, if not all,gp120 sequences preserve the virus's ability to bind to the viralreceptor CD4.

Env polypeptides (e.g., gp120, gp140 and gp160) include a “bridgingsheet” comprised of 4 anti-parallel β-strands (β-2, β-3, β-20 and β-21)that form a 3-sheet. Extruding from one pair of the β-strands (β-2 andβ-3) are two loops, V1 and V2. The β-2 sheet occurs at approximatelyamino acid residue 113 (Cys) to amino acid residue 117 (Thr) while β-3occurs at approximately amino acid residue 192 (Ser) to amino acidresidue 194 (Ile), relative to SF-162. The “V1/V2 region” occurs atapproximately amino acid positions 120 (Cys) to residue 189 (Cys),relative to SF-162. (see, e.g., Wyatt et al. (1995) J. Virol.69:5723-5733; Stamatatos et al. (1998) J. Virol. 72:7840-7845).Extruding from the second pair of β-strands (β-20 and β-21) is a“small-loop” structure, also referred to herein as “the bridging sheetsmall loop.” The locations of both the small loop and bridging sheetsmall loop can be determined relative to HXB-2 following the teachingsherein and in WO00/39303. Table 1 provides a list of synthetic genesencoding representative Env polypeptide based upon the SF162 strain andthe corresponding SEQ ID NOs.

TABLE 1 Exemplary Synthetic Env Polypeptide Expression Cassettes (SF162)Expression Cassette Seq Id Description gp120 SF162 1 wild-type gp140SF162 2 wild-type gp160 SF162 3 wild-type gp120.modSF162 4 nonegp120.modSF162.delV2 5 deleted V2 loop gp120.modSF162.delV1/V2 6 deletedV1 and V2 gp140.modSF162 7 none gp140.modSF162.delV2 8 deleted V2 loopgp140.modSF162.delVl/V2 9 deleted V1 and V2 gp140.mut.modSF162 10mutated cleavage site gp140.mut.modSF162.delV2 11 deleted V2; mutatedcleavage site gp140.mut.modSF162.delV1/V2 12 deleted V1 & V2; mutatedcleavage site gp140.mut7.modSF162 13 mutated cleavage sitegp140.mut7.modSF162.delV2 14 mutated cleavage site; deleted V2gp140.mut7.modSF162.delV1/V2 15 mutated cleavage site; deleted V1 and V2gp140.mut8.modSF162 16 mutated cleavage site gp140.mut8.modSF162.delV217 mutated cleavage site; deletedV2 gp140.mut8.modSF162.delV1/V2 18mutated cleavage site; deleted V1 and V2 gp160.modSF162 19 nonegp160.modSF162.delV2 20 deleted V2 loop gp160.modSF162.delV1/V2 21deleted V1 & V2 TV1 polypeptide 22 SF162 polypeptide 23

Furthermore, Env polypeptides are not limited to a polypeptide havingone of the exact sequences described herein. Indeed, the HIV genome isin a state of constant flux and contains several variable domains whichexhibit relatively high degrees of variability between isolates. It isreadily apparent that the terms encompass Env (e.g., gp160, gp140, andgp120) polypeptides from any of the identified HIV isolates, as well asnewly identified isolates, and subtypes of these isolates. Descriptionsof structural features are given herein with reference to SF162. One ofordinary skill in the art in view of the teachings of the presentdisclosure and the art can determine corresponding regions in other HIVvariants (e.g., isolates HIV_(IIIb), HIV_(SF2), HIV-1_(SF162),HIV-1_(SF170), HIV_(LAV), HIV_(LAI), HIV_(MN), HIV-1_(CM235),HIV-1_(US4), other HIV-1 strains from diverse subtypes (e.g., subtypes,A through G, and O), HIV-2 strains and diverse subtypes (e.g.,HIV-2_(UC1) and HIV-2_(UC2)), and simian immunodeficiency virus (SIV).(See, e.g., Virology, 3rd Edition (W. K. Joklik ed. 1988); FundamentalVirology, 2nd Edition (B. N. Fields and D. M. Knipe, eds. 1991);Virology, 3rd Edition (Fields, B N, D M Knipe, P M Howley, Editors,1996, Lippincott-Raven, Philadelphia, Pa.; for a description of theseand other related viruses), using for example, sequence comparisonprograms (e.g., BLAST and others described herein) or identification andalignment of structural features (e.g., a program such as the “ALB”program described herein that can identify P-sheet regions). The actualamino acid sequences of the Env polypeptides can be based on any HIVvariant.

Additionally, the term Env polypeptide (e.g., gp160, gp140, and gp120)encompasses proteins which include additional modifications to thenative sequence, such as additional internal deletions, additions andsubstitutions. These modifications may be deliberate, as throughsite-directed mutagenesis, or may be accidental, such as throughnaturally occurring mutational events. However, the modifications mustbe such that immunological activity (i.e., the ability to elicit anantibody response to the Env polypeptides found in HIV) is not lost.

Examples of modifications and mutations to Env polypeptides includedeletions or replacements of all or a part of the bridging sheet portionand, optionally, the variable regions V1 and V2. Generally, modified Envpolypeptides have enough of the bridging sheet removed to expose the CD4binding site, but leave enough of the structure to allow correct folding(e.g., correct geometry). Thus, modifications to the β-20 and β-21regions (between about amino acid residues 420 and 435 relative toHXB-2) are preferred. Additionally, modifications to the β-2 and β-3regions (between about amino acid residues 119 (Cys) and 201 (Ile)) andmodifications (e.g., deletions) to the V1 and V2 loop regions may alsobe made. Other exemplary mutations can abrogate the cleavage site in Envto prevent enzymatic cleavage of oligomeric gp140 into gp120 monomers.(See, e.g., Earl et al. (1990) PNAS USA 87:648-652; Earl et al. (1991)J. Virol. 65:31-41). In yet other embodiments, N-glycosylation sites maybe removed. Additional modifications and mutations to Env polypeptidesmay be found in WO00/39303, WO00/39302, WO00/39304, and WO02/04493.Additional examples of Env polypeptides may be found in U.S. Pat. No.5,792,459 (for a description of HIV_(SF2) Env polypeptides).

An immunogenic Env polypeptide is a molecule that includes at least oneepitope such that the molecule is capable of either eliciting animmunological reaction in an individual to which the protein isadministered or, in the diagnostic context, is capable of reacting withantibodies directed against the HIV in question.

Additional HIV Polypeptides

Wild-type HIV coding sequences for additional HIV polypeptides (e.g.,Gag, Pol, tat, rev, nef, vpr, vpu, vif, etc.) can be selected from anyknown HIV isolate. The wild-type coding region may be modified in anyway including one or more of the ways discussed below. As discussedabove, different mutations may be introduced into the coding sequencesof different genes.

The HIV genome and various polypeptide-encoding regions are shown inTable 2. The nucleotide positions are given relative to 8-5_TV1_C.ZA (anHIV Type C isolate). However, it will be readily apparent to one ofordinary skill in the art in view of the teachings of the presentdisclosure how to determine corresponding regions in other HIV strainsor variants (e.g., isolates HIV_(IIIb), HIV_(SF2), HIV-1_(SF162),HIV-1_(SF170), HIV_(LAV), HIV_(LAI), HIV_(MN), HIV-1_(CM235),HIV-1_(US4)) other HIV-1 strains from diverse subtypes (e.g., subtypes,A through G, and O), HIV-2 strains and diverse subtypes (e.g., HIV-2UC1and HIV-2UC2), and simian immunodeficiency virus (SIV). (See, e.g.,Virology, 3rd Edition (W. K. Joklik ed. 1988); Fundamental Virology, 2ndEdition (B. N. Fields and D. M. Knipe, eds. 1991); Virology, 3rd Edition(Fields, B N, D M Knipe, P M Howley, Editors, 1996, Lippincott-Raven,Philadelphia, Pa.; for a description of these and other relatedviruses), using for example, sequence comparison programs (e.g., BLASTand others described herein) or identification and alignment ofstructural features (e.g., a program such as the “ALB” program describedherein that can identify the various regions).

TABLE 2 Regions of the HIV Genome relative to 8_5_TV1_C.ZA RegionPosition in nucleotide sequence 5′LTR  1-636 U3  1-457 R 458-553 U5554-636 NFkB II 340-348 NFkB I 354-362 Sp1 III 379-388 Sp1 II 390-398Sp1 I 400-410 TATA Box 429-433 TAR 474-499 Poly A signal 529-534 PBS638-655 p7 binding region, packaging signal 685-791 Gag:  792-2285 p17 792-1178 p24 1179-1871 Cyclophilin A bdg. 1395-1505 MHR 1632-1694 p21872-1907 p7 1908-2072 Frameshift slip 2072-2078 p1 2073-2120 p6Gag2121-2285 Zn-motif I 1950-1991 Zn-motif II 2013-2054 Pol: 2072-5086p6Pol 2072-2245 Prot 2246-2542 p66RT 2543-4210 p15RNaseH 3857-4210p31Int 4211-5086 Vif: 5034-5612 Hydrophilic region 5292-5315 Vpr:5552-5839 Oligomerization 5552-5677 Amphipathic a-helix 5597-5653 Tat:5823-6038 and 8417-8509 Tat-1 exon 5823-6038 Tat-2 exon 8417-8509N-terminal domain 5823-5885 Trans-activation domain 5886-5933Transduction domain 5961-5993 Rev: 5962-6037 and 8416-8663 Rev-1 exon5962-6037 Rev-2 exon 8416-8663 High-affinity bdg. site 8439-8486Leu-rich effector domain 8562-8588 Vpu: 6060-6326 Transmembrane domain6060-6161 Cytoplasmic domain 6162-6326 Env (gp160): 6244-8853 Signalpeptide 6244-6324 gp120 6325-7794 V1 6628-6729 V2 6727-6852 V3 7150-7254V4 7411-7506 V5 7663-7674 C1 6325-6627 C2 6853-7149 C3 7255-7410 C47507-7662 C5 7675-7794 CD4 binding 7540-7566 gp41 7795-8853 Fusionpeptide 7789-7842 Oligomerization domain 7924-7959 N-terminal heptadrepeat 7921-8028 C-terminal heptad repeat 8173-8280 Immunodominantregion 8023-8076 Nef: 8855-9478 Myristoylation 8858-8875 SH3 binding9062-9091 Polypurine tract 9128-9154 SH3 binding 9296-9307

Gag Polypeptides

The additional HIV polypeptides may include Gag polypeptides. The fulllength Gag-polymerase sequence may be included in the Gag polypeptide inorder to increase the number of epitopes. Because such full lengthpolypeptides include the potentially deleterious functional enzymesreverse transcriptase (RT) and integrase (INT) (in addition to thestructural proteins and protease), it is important to inactivate RT andINT functions. Several in-frame deletions in the RT and INT readingframe can be made to achieve catalytic nonfunctional enzymes withrespect to their RT and INT activity. (See, e.g., Jay. A. Levy (Editor)(1995) The Retroviridae, Plenum Press, New York. ISBN 0-306-45033×.Pages 215-20; Grimison, B. and Laurence, J. (1995), Journal Of AcquiredImmune Deficiency Syndromes and Human Retrovirology 9(1):58-68;Wakefield, J. K., et al., (1992) Journal Of Virology 66(11):6806-6812;Esnouf, R., et al., (1995) Nature Structural Biology 2(4):303-308;Maignan, S., et al., (1998) Journal Of Molecular Biology 282(2):359-368;Katz, R. A. and Skalka, A. M. (1994) Annual Review Of Biochemistry 73(1994); Jacobo-Molina, A., et al., (1993) Proceedings Of the NationalAcademy Of Sciences Of the United States Of America 90(13):6320-6324;Hickman, A. B., et al., (1994) Journal Of Biological Chemistry269(46):29279-29287; Goldgur, Y., et al., (1998) Proceedings Of theNational Academy Of Sciences Of the United States Of America95(16):9150-9154; Goette, M., et al., (1998) Journal Of BiologicalChemistry 273(17):10139-10146; Gorton, J. L., et al., (1998) Journal ofVirology 72(6):5046-5055; Engelman, A., et al., (1997) Journal OfVirology 71(5):3507-3514; Dyda, F., et al., Science 266(5193):1981-1986; Davies, J. F., et al., (1991) Science 252(5002):88-95;Bujacz, G., et al., (1996) Febs Letters 398(2-3):175-178; Beard, W. A.,et al., (1996) Journal Of Biological Chemistry 271(21):12213-12220;Kohlstaedt, L. A., et al., (1992) Science 256(5065):1783-1790; Krug, M.S. and Berger, S. L. (1991) Biochemistry 30(44):10614-10623; Mazumder,A., et al., (1996) Molecular Pharmacology 49(4):621-628; Palaniappan,C., et al., (1997) Journal Of Biological Chemistry 272(17):11157-11164;Rodgers, D. W., et al., (1995) Proceedings Of the National Academy OfSciences Of the United States Of America 92(4):1222-1226; Sheng, N. andDennis, D. (1993) Biochemistry 32(18):4938-4942; Spence, R. A., et al.,(1995) Science 267(5200):988-993.}

Furthermore selected B- and/or T-cell epitopes can be added to theGag-polymerase polypeptides within the deletions of the RT- andINT-coding sequence to replace and augment any epitopes deleted by thefunctional modifications of RT and INT. Alternately, selected B- andT-cell epitopes (including CTL epitopes) from RT and INT can be includedin other additional HIV polypeptides. (For descriptions of known HIV B-and T-cell epitopes see, HIV Molecular Immunology Database CTL SearchInterface; Los Alamos Sequence Compendia, 1987-1997; Internet addresshiv-web.lan1.gov under the directory immunology/index.html).

Representative mutations to the protease include attenuation of proteaseactivity (Thr26Ser) and inactivation of the protease (Asp25Ala) (e.g.,Konvalinka et al., 1995, J Virol 69:7180-86). Representative mutationsto the reverse transcriptase include deletion of the catalytic center(e.g., Biochemistry, 1995, 34, 5351, Patel et al.), and deletion of theprimer grip region (e.g., J Biol Chem, 272, 17, 11157, Palaniappan, etal., 1997). Representative mutations to the integrase include mutationof the HHCC domain (Cys40Ala), inactivation of the catalytic center(Asp64Ala, Aspl 16Ala, Glu 152Ala) (e.g., Wiskerchen et al., 1995, JVirol, 69:376), and inactivation of the minimal DNA binding domain(MDBD) (deletion of Trp235) (e.g., Ishikawa et al., 1999, J Virol, 73:4475).

Pol Polypeptides

The additional HIV polypeptides may include Pol polypeptides. Polpolypeptides include, but are not limited to, the protein-encodingregions comprising polymerase, protease, reverse transcriptase and/orintegrase-containing sequences (Wan et al. (1996) Biochem. J.316:569-573; Kohl et al. (1988) PNAS USA 85:4686-4690; Krausslich et al.(1988) J. Virol. 62:4393-4397; Coffin, “Retroviridae and theirReplication” in Virology, pp 1437-1500 (Raven, New York, 1990); Patel etal. (1995) Biochemistry 34:5351-5363). Thus, the Pol polypeptides hereininclude one or more of these regions and one or more changes to theresulting amino acid sequences.

In certain embodiments, the catalytic center and/or primer grip regionof RT are modified as described above. The catalytic center and primergrip regions of RT are described, for example, in Patel et al. (1995)Biochem. 34:5351 and Palaniappan et al. (1997) J. Biol. Chem. 272(17):11157. For example, wild type sequence encoding the amino acids YMDD atpositions 183-185 of p66 RT, numbered relative to AF110975, may bereplaced with sequence encoding the amino acids “AP”. Further, theprimer grip region (amino acids WMGY, residues 229-232 of p66RT,numbered relative to AF110975) may be replaced with sequence encodingthe amino acids “PI.”

Vif, Vpr, and Vpu Polypeptides

The additional HIV polypeptides may include Vif, Vpr and Vpupolypeptides. Reducing or eliminating the function of the associatedgene products can be accomplished employing routine methods available inthe art. By way of example, Simon et al. (J. Virol 73:2675-81, 1999)teach mutations of Vif. Simon et al. (J. Virol. 74:10650-57, 2000) teachmutations of Vpr. Tiganos et al. (Virology 251:96-107, 1998) teachmutation of Vpu.

Tat Polypeptides

The additional HIV polypeptides may include Tat polypeptides. Tatpolypeptides may be modified using routine methods taught in the art(e.g., replacing a cysteine residue at position 22 with a glycine or acysteine at position 37 with a serine, Caputo et al. Gene Therapy 3:235,1996).

Rev Polypeptides

The additional HIV polypeptides may include Rev polypeptides. Revpolypeptides may be modified using routine methods taught in the art(e.g., mutations in the Rev domains (e.g., Thomas et al., 1998, J Virol.72: 2935-44), mutation in RNA binding-nuclear localization(ArgArg38,39AspLeu=M5), and mutation in the activation domain(LeuGlu78,79AspLeu=M10)).

Nef Polypeptides

The additional HIV polypeptides may include Nefpolypeptides. Nefpolypeptides may be modified using routine methods taught in the art(e.g., mutations of the myristoylation signal and in the oligomerizationdomain: point mutations to the myristoylation signal (Gly-to-Ala=−Myr),deletion of N-terminal first 18 (sub-type B, e.g., SF162) or 19(sub-type C, e.g., South Africa clones) amino acids: −Myr18 or −Myr19(respectively) (e.g., Peng and Robert-Guroff, 2001, Immunol Letters 78:195-200), single point mutation to the oligomerization domain (Asp125Gly(sub B SF162) or Asp 124Gly (sub C South Africa clones)) (e.g., Liu etal., 2000, J Virol 74: 5310-19), and mutations affecting (1) infectivity(replication) of HIV-virions and/or (2) CD4 down regulation. (e.g.,Lundquist et al. (2002) J Virol. 76(9): 4625-33)).

Methods of Producing Env Polypeptides and Additional HIV Polypeptides

The polypeptides disclosed herein can be produced in any number of wayswhich are well known in the art.

In one embodiment, the polypeptides are generated using recombinanttechniques, well known in the art. In this regard, oligonucleotideprobes can be devised based on the known sequences of Env and other HIVpolypeptides and used to probe genomic or cDNA libraries for Env andother HIV genes. The gene can then be further isolated using standardtechniques, e.g., restriction enzymes employed to truncate the gene atdesired portions of the full-length sequence. Similarly, Env and otherHIV genes can be isolated directly from cells and tissues containing thesame, using known techniques, such as phenol extraction and the sequencefurther manipulated to produce the desired truncations. See, e.g.,Sambrook et al., supra, for a description of techniques used to obtainand isolate DNA.

The genes encoding the modified (e.g., truncated and/or substituted)polypeptides can be produced synthetically, based on the knownsequences. The nucleotide sequence can be designed with the appropriatecodons for the particular amino acid sequence desired. The completesequence is generally assembled from overlapping oligonucleotidesprepared by standard methods and assembled into a complete codingsequence. See, e.g., Edge (1981) Nature 292:756; Nambair et al. (1984)Science 223:1299; Jay et al. (1984) J. Biol. Chem. 259:6311; Stemmer etal. (1995) Gene 164:49-53.

Recombinant techniques are readily used to clone a gene encoding Env andother HIV polypeptide genes which can then be mutagenized in vitro bythe replacement of the appropriate base pair(s) to result in the codonfor the desired amino acid. Such a change can include as little as onebase pair, effecting a change in a single amino acid, or can encompassseveral base pair changes. Alternatively, the mutations can be effectedusing a mismatched primer which hybridizes to the parent nucleotidesequence (generally cDNA corresponding to the RNA sequence), at atemperature below the melting temperature of the mismatched duplex. Theprimer can be made specific by keeping primer length and basecomposition within relatively narrow limits and by keeping the mutantbase centrally located. See, e.g., Innis et al., (1990) PCRApplications: Protocols for Functional Genomics; Zoller and Smith,Methods Enzymol. (1983) 100:468. Primer extension is effected using DNApolymerase; the product cloned and clones containing the mutated DNA,derived by segregation of the primer extended strand, selected.Selection can be accomplished using the mutant primer as a hybridizationprobe. The technique is also applicable for generating multiple pointmutations. See, e.g., Dalbie-McFarland et al. Proc. Natl. Acad. Sci. USA(1982) 79:6409.

Once coding sequences for the desired proteins have been isolated orsynthesized, they can be cloned into any suitable vector or replicon forexpression. As will be apparent from the teachings herein, a widevariety of vectors encoding modified polypeptides can be generated bycreating expression constructs which operably link, in variouscombinations, polynucleotides encoding Env and other HIV polypeptideshaving deletions or mutation therein. Thus, for example, polynucleotidesencoding a particular portion with the deleted V1/V2 region for an Envpolypeptide can be operably linked with polynucleotides encoding Envpolypeptides having deletions or replacements in the small loop regionand the construct introduced into a host cell for expression of the Envpolypeptide.

Numerous cloning vectors are known to those of skill in the art, and theselection of an appropriate cloning vector is a matter of choice.Examples of recombinant DNA vectors for cloning and host cells whichthey can transform include the bacteriophage lambda (E. coli), pBR322(E. coli), pACYC177 (E. coli), pKT230 (gram-negative bacteria), pGV1106(gram-negative bacteria), pLAFR1 (gram-negative bacteria), pME290(non-E. coli gram-negative bacteria), pHV14 (E. coli and Bacillussubtilis), pBD9 (Bacillus), pIJ61 (Streptomyces), pUC6 (Streptomyces),YIp5 (Saccharomyces), YCp19 (Saccharomyces) and bovine papilloma virus(mammalian cells). See, generally, DNA Cloning: Vols. I & II, supra;Sambrook et al., supra; B. Perbal, supra.

Insect cell expression systems, such as baculovirus systems, can also beused and are known to those of skill in the art and described in, e.g.,Summers and Smith, Texas Agricultural Experiment Station Bulletin No.1555 (1987). Materials and methods for baculovirus/insect cellexpression systems are commercially available in kit form from, interalia, Invitrogen, San Diego Calif. (“MaxBac” kit).

Plant expression systems can also be used to produce Env and other HIVpolypeptides. Generally, such systems use virus-based vectors totransfect plant cells with heterologous genes. For a description of suchsystems see, e.g., Porta et al., Mol. Biotech. (1996) 5:209-221; andHackland et al., Arch. Virol. (1994) 139:1-22.

Viral systems, such as a vaccinia based infection/transfection system,as described in Tomei et al., J. Virol. (1993) 67:4017-4026 and Selby etal., J. Gen. Virol. (1993) 74:1103-1113, will also find use with thepresent invention. In this system, cells are first transfected in vitrowith a vaccinia virus recombinant that encodes the bacteriophage T7 RNApolymerase. This polymerase displays exquisite specificity in that itonly transcribes templates bearing T7 promoters. Following infection,cells are transfected with the DNA of interest, driven by a T7 promoter.The polymerase expressed in the cytoplasm from the vaccinia virusrecombinant transcribes the transfected DNA into RNA which is thentranslated into protein by the host translational machinery. The methodprovides for high level, transient, cytoplasmic production of largequantities of RNA and its translation product(s).

The gene can be placed under the control of a promoter, ribosome bindingsite (for bacterial expression) and, optionally, an operator(collectively referred to herein as “control” elements), so that the DNAsequence encoding the desired Env or other HIV polypeptide istranscribed into RNA in the host cell transformed by a vector containingthis expression construction. The coding sequence may or may not containa signal peptide or leader sequence. Both the naturally occurring signalpeptides and heterologous sequences can be used. Leader sequences can beremoved by the host in post-translational processing. See, e.g., U.S.Pat. Nos. 4,431,739; 4,425,437; 4,338,397. Such sequences include, butare not limited to, the TPA leader, as well as the honey bee mellitinsignal sequence.

Other regulatory sequences may also be desirable which allow forregulation of expression of the protein sequences relative to the growthof the host cell. Such regulatory sequences are known to those of skillin the art, and examples include those which cause the expression of agene to be turned on or off in response to a chemical or physicalstimulus, including the presence of a regulatory compound. Other typesof regulatory elements may also be present in the vector, for example,enhancer sequences.

The control sequences and other regulatory sequences may be ligated tothe coding sequence prior to insertion into a vector. Alternatively, thecoding sequence can be cloned directly into an expression vector whichalready contains the control sequences and an appropriate restrictionsite.

In some cases it may be necessary to modify the coding sequence so thatit may be attached to the control sequences with the appropriateorientation; i.e., to maintain the proper reading frame. Mutants oranalogs may be prepared by the deletion of a portion of the sequenceencoding the protein, by insertion of a sequence, and/or by substitutionof one or more nucleotides within the sequence. Techniques for modifyingnucleotide sequences, such as site-directed mutagenesis, are well knownto those skilled in the art. See, e.g., Sambrook et al., supra; DNACloning, Vols. I and II, supra; Nucleic Acid Hybridization, supra.

The expression vector is then used to transform an appropriate hostcell. A number of mammalian cell lines are known in the art and includeimmortalized cell lines available from the American Type CultureCollection (ATCC), such as, but not limited to, Chinese hamster ovary(CHO) cells, HeLa cells, baby hamster kidney (BHK) cells, monkey kidneycells (COS), human hepatocellular carcinoma cells (e.g., Hep G2),Vero293 cells, as well as others. For the Env polypeptides, expressionin mammalian cells is preferred to ensure proper glycosylation.Similarly, bacterial hosts such as E. coli, Bacillus subtilis, andStreptococcus spp., will find use with the present expressionconstructs. Yeast hosts useful in the present invention include interalia, Saccharomyces cerevisiae, Candida albicans, Candida maltosa,Hansenula polymorpha, Kluyveromyces fragilis, Kluyveromyces lactis,Pichia guillerimondii, Pichia pastoris, Schizosaccharomyces pombe andYarrowia lipolytica. Insect cells for use with baculovirus expressionvectors include, inter alia, Aedes aegypti, Bombyx mori, Drosophilamelanogaster, Spodoptera frugiperda, and Trichoplusia ni.

Depending on the expression system and host selected, the proteins ofthe present invention are produced by growing host cells transformed byan expression vector described above under conditions whereby theprotein of interest is expressed. The selection of the appropriategrowth conditions is within the skill of the art.

In one embodiment, the transformed cells secrete the polypeptide productinto the surrounding media. Certain regulatory sequences can be includedin the vector to enhance secretion of the protein product, for exampleusing a tissue plasminogen activator (TPA) leader sequence, aγ-interferon signal sequence or other signal peptide sequences fromknown secretory proteins. The secreted polypeptide product can then beisolated by various techniques described herein, for example, usingstandard purification techniques such as but not limited to,hydroxyapatite resins, column chromatography, ion-exchangechromatography, size-exclusion chromatography, electrophoresis, HPLC,immunoadsorbent techniques, affinity chromatography,immunoprecipitation, and the like.

Alternatively, the transformed cells are disrupted, using chemical,physical or mechanical means, which lyse the cells yet keep the Env orother HIV polypeptides substantially intact. Intracellular proteins canalso be obtained by removing components from the cell wall or membrane,e.g., by the use of detergents or organic solvents, such that leakage ofEnv or other HIV polypeptides occurs. Such methods are known to those ofskill in the art and are described in, e.g., Protein PurificationApplications: A Practical Approach, (E. L. V. Harris and S. Angal, Eds.,1990)

For example, methods of disrupting cells for use with the presentinvention include but are not limited to: sonication or ultrasonication;agitation; liquid or solid extrusion; heat treatment; freeze-thaw;desiccation; explosive decompression; osmotic shock; treatment withlytic enzymes including proteases such as trypsin, neuraminidase andlysozyme; alkali treatment; and the use of detergents and solvents suchas bile salts, sodium dodecylsulphate, Triton, NP40 and CHAPS. Theparticular technique used to disrupt the cells is largely a matter ofchoice and will depend on the cell type in which the polypeptide isexpressed, culture conditions and any pre-treatment used.

Following disruption of the cells, cellular debris is removed, generallyby centrifugation, and the intracellularly produced Env and other HIVpolypeptides are further purified, using standard purificationtechniques such as but not limited to, column chromatography,ion-exchange chromatography, size-exclusion chromatography,electrophoresis, HPLC, immunoadsorbent techniques, affinitychromatography, immunoprecipitation, and the like.

For example, one method for obtaining intracellular Env polypeptides ofthe present invention involves affinity purification, such as byimmunoaffinity chromatography using anti-Env specific antibodies, or bylectin affinity chromatography. Particularly preferred lectin resins arethose that recognize mannose moieties such as but not limited to resinsderived from Galanthus nivalis agglutinin (GNA), Lens culinarisagglutinin (LCA or lentil lectin), Pisum sativum agglutinin (PSA or pealectin), Narcissus pseudonarcissus agglutinin (NPA) and Allium ursinumagglutinin (AUA). The choice of a suitable affinity resin is within theskill in the art. After affinity purification, the Env and other HIVpolypeptides can be further purified using conventional techniques wellknown in the art, such as by any of the techniques described above.

Relatively small polypeptides, i.e., up to about 50 amino acids inlength, can be conveniently synthesized chemically, for example by anyof several techniques that are known to those skilled in the peptideart. In general, these methods employ the sequential addition of one ormore amino acids to a growing peptide chain. Normally, either the aminoor carboxyl group of the first amino acid is protected by a suitableprotecting group. The protected or derivatized amino acid can then beeither attached to an inert solid support or utilized in solution byadding the next amino acid in the sequence having the complementary(amino or carboxyl) group suitably protected, under conditions thatallow for the formation of an amide linkage. The protecting group isthen removed from the newly added amino acid residue and the next aminoacid (suitably protected) is then added, and so forth. After the desiredamino acids have been linked in the proper sequence, any remainingprotecting groups (and any solid support, if solid phase synthesistechniques are used) are removed sequentially or concurrently, to renderthe final polypeptide. By simple modification of this general procedure,it is possible to add more than one amino acid at a time to a growingchain, for example, by coupling (under conditions which do not racemizechiral centers) a protected tripeptide with a properly protecteddipeptide to form, after deprotection, a pentapeptide. See, e.g., J. M.Stewart and J. D. Young, Solid Phase Peptide Synthesis (Pierce ChemicalCo., Rockford, Ill. 1984) and G. Barany and R. B. Merrifield, ThePeptides: Analysis, Synthesis, Biology, editors E. Gross and J.Meienhofer, Vol. 2, (Academic Press, New York, 1980), pp. 3-254, forsolid phase peptide synthesis techniques; and M. Bodansky, Principles ofPeptide Synthesis, (Springer-Verlag, Berlin 1984) and E. Gross and J.Meienhofer, Eds., The Peptides: Analysis, Synthesis, Biology, Vol. 1,for classical solution synthesis.

Typical protecting groups include t-butyloxycarbonyl (Boc),9-fluorenylmethoxycarbonyl (Fmoc) benzyloxycarbonyl (Cbz);p-toluenesulfonyl (Tx); 2,4-dinitrophenyl; benzyl (Bzl);biphenylisopropyloxycarboxy-carbonyl, t-amyloxycarbonyl,isobornyloxycarbonyl, o-bromobenzyloxycarbonyl, cyclohexyl, isopropyl,acetyl, o-nitrophenylsulfonyl and the like.

Typical solid supports are cross-linked polymeric supports. These caninclude divinylbenzene cross-linked-styrene-based polymers, for example,divinylbenzene-hydroxymethylstyrene copolymers,divinylbenzene-chloromethylstyrene copolymers anddivinylbenzene-benzhydrylaminopolystyrene copolymers.

The polypeptide analogs of the present invention can also be chemicallyprepared by other methods such as by the method of simultaneous multiplepeptide synthesis. See, e.g., Houghten Proc. Natl. Acad. Sci. USA (1985)82:5131-5135; U.S. Pat. No. 4,631,211.

Vaccines

The Env polypeptides complexed to polyanionic carbomers and immunogeniccompositions comprising such complexes (“Env polypeptide complexes”) andthe Env polypeptides with low viscosity, polyanionic polymers (Envpolypeptide complexes and low viscosity, polyanionic carbomer-Envpolypeptide compositions collectively are “Env compositions”) can beused in various vaccine compositions, individually or in combination, ine.g., prophylactic (i.e., to prevent infection) or therapeutic (to treatHIV following infection) vaccines. The vaccines can comprise mixtures ofone or more Env polypeptides, such as Env polypeptides derived from morethan one viral isolate. The vaccine may also be administered inconjunction with other antigens and immunoregulatory agents, forexample, immunoglobulins, cytokines, lymphokines, and chemokines,including but not limited to IL-2, modified IL-2 (cys125→ser125),GM-CSF, IL-12, γ-interferon, IP-10, MIP1β and RANTES. The vaccines mayalso comprise a mixture of protein and nucleic acid, which in turn maybe delivered using the same or different vehicles. The Env compositionvaccines may be given more than once (e.g., a “prime” administrationfollowed by one or more “boosts”) to achieve the desired effects. Thesame composition can be administered as the prime and as the one or moreboosts. Alternatively, different compositions can be used for primingand boosting.

By way of example, any of the Env composition vaccines can be used incombination with other DNA delivery systems and/or protein deliverysystems with HIV antigens. Non-limiting examples includeco-administration of these molecules, for example, in prime-boostmethods where one or more molecules are delivered in a “priming” stepand, subsequently, one or more molecules are delivered in a “boosting”step. In certain embodiments, the delivery of one or more nucleicacid-containing compositions and is followed by delivery of the Envcomposition vaccines. In other embodiments, multiple nucleic acid“primes” (of the same or different nucleic acid molecules) can befollowed by multiple Env composition “boosts” (of the same or differentEnv polypeptides and additional HIV polypeptides).

The vaccines will generally include one or more pharmaceuticallyacceptable excipients or vehicles such as water, saline, glycerol,ethanol, etc. Additionally, auxiliary substances, such as wetting oremulsifying agents, pH buffering substances, and the like, may bepresent in such vehicles.

A carrier is optionally present. Carriers are molecules that do notalone induce the production of antibodies harmful to the individualreceiving the composition. Suitable carriers are typically large, slowlymetabolized macromolecules such as proteins, polysaccharides, polylacticacids, polyglycolic acids, polymeric amino acids, amino acid copolymers,lipid aggregates (such as oil droplets or liposomes), and inactive virusparticles. Such carriers are well known to those of ordinary skill inthe art. Furthermore, the Env polypeptide in the Env compositions may beconjugated to a bacterial toxoid, such as toxoid from diphtheria,tetanus, cholera, etc.

Adjuvants may also be used to enhance the effectiveness of the vaccines.Such adjuvants include, but are not limited to: (1) aluminum salts(alum), such as aluminum hydroxide, aluminum phosphate, aluminumsulfate, etc.; (2) oil-in-water emulsion formulations (with or withoutother specific immunostimulating agents such as muramyl peptides (seebelow) or bacterial cell wall components), such as for example (a) MF59™ (International Publication No. WO 90/14837), containing 5% Squalene,0.5% TWEEN 80 ™, and 0.5% SPAN 85 ™ (optionally containing variousamounts of MTP-PE (see below), although not required) formulated intosubmicron particles using a microfluidizer such as Model 110Ymicrofluidizer (Microfluidics, Newton, Mass.), (b) SAF, containing 10%Squalane, 0.4% TWEEN 80™, 5% pluronic-blocked polymer L121, and thr-MDP(see below) either microfluidized into a submicron emulsion or vortexedto generate a larger particle size emulsion, and (c) RIBI™ adjuvantsystem (RAS), (Ribi Immunochem, Hamilton, Mont.) containing 2% Squalene,0.2% TWEEN 80™, and one or more bacterial cell wall components from thegroup consisting of monophosphorylipid A (MPL), trehalose dimycolate(TDM), and cell wall skeleton (CWS), preferably MPL+CWS (DETOX™); (3)saponin adjuvants, such as STIMULON™ (Cambridge Bioscience, Worcester,Mass.) may be used or particle generated therefrom such as ISCOMs(immunostimulating complexes); (4) Complete Freunds Adjuvant (CFA) andIncomplete Freunds Adjuvant (IFA); (5) cytokines, such as interleukins(IL-1, IL-2, etc.), macrophage colony stimulating factor (M-CSF), tumornecrosis factor (TNF), etc.; (6) detoxified mutants of a bacterialADP-ribosylating toxin such as a cholera toxin (CT), a pertussis toxin(PT), or an E. coli heat-labile toxin (LT), particularly LT-K63 (wherelysine is substituted for the wild-type amino acid at position 63)LT-R72 (where arginine is substituted for the wild-type amino acid atposition 72), CT-S109 (where serine is substituted for the wild-typeamino acid at position 109), and PT-K9/G129 (where lysine is substitutedfor the wild-type amino acid at position 9 and glycine substituted atposition 129) (see, e.g., International Publication Nos. WO93/13202 andWO92/19265); and (7) other substances that act as immunostimulatingagents to enhance the effectiveness of the composition.

Typically, the vaccine compositions are prepared as injectables, eitheras liquid solutions or suspensions; solid forms suitable for solutionin, or suspension in, liquid vehicles prior to injection may also beprepared. The preparation also may be emulsified or encapsulated inliposomes for enhanced adjuvant effect, as discussed above.

The vaccines will comprise a therapeutically effective amount of the Envcompositions and any other of the above-mentioned components, as needed.A therapeutically effective amount will be an amount of the Envcomposition that will induce a protective immunological response in theuninfected, infected or unexposed individual to which it isadministered. Such a response will generally result in the developmentin the subject of a secretory, cellular and/or antibody-mediated immuneresponse to the vaccine. Usually, such a response includes but is notlimited to one or more of the following effects; the production ofantibodies from any of the immunological classes, such asimmunoglobulins A, D, E, G or M; the proliferation of B and Tlymphocytes; the provision of activation, growth and differentiationsignals to immunological cells; expansion of helper T cell, suppressor Tcell, and/or cytotoxic T cell.

Preferably, the effective amount is sufficient to bring about treatmentor prevention of disease symptoms. The exact amount necessary will varydepending on the subject being treated; the age and general condition ofthe individual to be treated; the capacity of the individual's immunesystem to synthesize antibodies; the degree of protection desired; theseverity of the condition being treated; the particular Env polypeptideselected and its mode of administration, among other factors. Anappropriate effective amount can be readily determined by one of skillin the art. A therapeutically effective amount will fall in a relativelybroad range that can be determined through routine trials.

The Env composition vaccines can be injected either subcutaneously,epidermally, intradermally, intramucosally such as nasally, rectally andvaginally, intraperitoneally, intravenously, orally or intramuscularly.Other modes of administration include oral and pulmonary administration,suppositories, needle-less injection, transcutaneous and transdermalapplications. Dosage treatment may be a single dose schedule or amultiple dose schedule.

General

The term “comprising” encompasses “including” as well as “consisting”,e.g., a composition “comprising” X may consist exclusively of X or mayinclude something additional, e.g., X+Y.

The word “substantially” does not exclude “completely”, e.g., acomposition which is “substantially free” from Y may be completely freefrom Y. Where necessary, the word “substantially” may be omitted fromthe definition of the invention. The term “about” in relation to anumerical value x means, for example, x±10%.

Unless specifically stated, a process comprising a step of mixing two ormore components does not require any specific order of mixing. Thuscomponents can be mixed in any order. Where there are three componentsthen two components can be combined with each other, and then thecombination may be combined with the third component, etc.

Where animal (and particularly bovine) materials are used in the cultureof cells, they should be obtained from sources that are free fromtransmissible spongiform encephalopathies (TSEs), and in particular freefrom bovine spongiform encephalopathy (BSE). Overall, it is preferred toculture cells in the total absence of animal-derived materials.

Where a cell substrate is used for reassortment or reverse geneticsprocedures, it is preferably one that has been approved for use in humanvaccine production, e.g., as in Ph Eur general chapter 5.2.3.

Identity between polypeptide sequences is preferably determined by theSmith-Waterman homology search algorithm as implemented in the MPSRCHprogram (Oxford Molecular), using an affine gap search with parametersgap open penalty=12 and gap extension penalty=1.

As used in this specification, the singular forms “a,” “an” and “the”include plural references unless the content clearly dictates otherwise.Thus, for example, reference to “an antigen” includes a mixture of twoor more such agents.

EXAMPLES Example 1 Generation of Polyanionic Carbomer+Env Complexes

CARBOPOL 971P NF™ was weighed under sterile condition. Half the finalvolume of 0.2μ filtered, distilled H₂O was added to the CARBOPOL™ powderand left in a rotator for end-over-end mixing for 5-10 minutes. Theremaining volume of water was then added and left in rotator forend-over-end mixing for 16-18 hours to allow a uniform suspension toform. Using these methods, homogeneous suspensions of 1-2% CARBOPOL™ canbe readily made. Longer periods of continuous mixing were required forhigher concentrations of CARBOPOL™ to ensure a homogenous suspension.Suspensions above 4% took longer to form and were too viscous to handlefor analytical or gel analysis purposes after formation of the Envcomplexes. The pH of the final solution was measured and typically foundin the range of pH 3.0-4.0. Due to high viscosity, suspensions ofgreater than 2% Carbopol were not tested in any in vitro or in vivoapplications.

The pH was important for the formation of the complexes. When the pH wasadjusted to 7.0 by addition of 3M NaOH/1M KOH before addition of the Envpolypeptide, Dynamic Light Scattering (DLS) analysis showed nointeraction between the Env polypeptide and the CARBOPOL™. By contrast,when the Env polypeptide was added to the low pH (3.0-4.0) acidicCARBOPOL™, the Env polypeptides and the CARBOPOL™ formed a complex,predominantly mediated by electrostatic interactions. At pH 3.0-4.0, Envpolypeptide is positively charged while polyanionic carbomers such asCARBOPOL™ are negatively charged—this allows charged-charged interactionbetween Env polypeptide and polyanionic molecules or polymers,facilitating the formation of complex. If CARBOPOL™ or similarpolyanionic carbomers are first adjusted to pH ˜7, then directinteraction with Env polypeptide (or with other positively chargedproteins) will not occur and hence no complexation. With the correct pH,the complexes form relatively quickly, requiring only incubation for ≦1hour. The complexes form readily, possibly much sooner than 1 hr sincecharge-charge interactions are instantaneous and rapid.

Example 2 Stability of the Env Polypeptide Complexed with AnionicCarbomers

In attempting to stabilize soluble, recombinant Env polypeptides forvaccination and to increase the adjuvantation provided by adjuvants suchas MF59™, a polyanionic carbomer, CARBOPOL 971P NF™, was tested. Toassess the stability of Env polypeptides complexed with polyanioniccarbomers, purified oligomer SF162 gp140ΔV2 Env polypeptide producedfrom CHO cells complexed with 0.5% CARBOPOL 971P NF™ was incubated forvarying time-periods at 4° C. and analyzed by SDS-PAGE andimmunoblotting, using anti-gp120 rabbit polyclonal sera. As shown inFIG. 1(A), the CARBOPOL 971P NF™ had no detrimental effect on Env. TheEnv polypeptide was stable in CARBOPOL 971P NF™-Env polypeptide complexat the longest time tested (4 hrs).

The stability of the Env polypeptide complexes was also tested atvarying temperatures: 1 hour at each of 4° C., room temperature (RT, 20°C.), 30° C. and 37° C. As a control, the Env polypeptide in PBS was alsoincubated at RT and 37° C. As shown in FIG. 1(B), the Env polypeptidecomplexes were stable for 1 hour at all temperatures (4° C., roomtemperature, 30° C. and 37° C.) tested. A faint band can be seen in lane4 of FIG. 1(B) at ˜70 kDa. This band corresponds to a known cleavageproduct of gp 140, generated by enzymatic cleavage of V3—loop; thisfragment has nothing to do with CARBOMER 971P NF™. We observed this athigher temperature, and not at lower temperature. This was expectedbecause the endoproteases that causes cleavage are active at thesetemperatures (room temperature to 37° C.). The Env polypeptide alone,therefore, was already showing some degree of degradation at one of thetwo temperatures tested—this again confirms that the enzymatic cleavageobserved here was due to enzymatic activity as favorable temperatures(RT-37° C.), and not specific to presence of CARBOMER 971P NF™. Incontrast, the Env polypeptide in complex with a polyanionic carbomer(CARBOMER 971P NF™) showed no degradation at any temperature tested,possibly also indicating that the protein can be partly protected fromenzymatic cleavage in this complex; the protection could be due to thelow pH (unfavorable for activity), steric hindrance caused by thepolyanionic carbomer interfering with the enzyme, or both. Thus, forminga complex stabilizes the Env polypeptide.

Example 3 Interaction of Env Polypeptide and Polyanionic Carbomers UsingDynamic Light Scattering (DLS)

To demonstrate that the Env polypeptide was forming a complex with thepolyanionic carbomer via direct interaction and thereby stabilizing theEnv polypeptide and/or aiding in the controlled release or presentationof the antigen, the complexes were analyzed by Dynamic Light Scattering(DLS) which measures the hydrodynamic radius of particles in solution.As shown in FIG. 2, the hydrodynamic radius of 0.5% CARBOPOL 971P NF™alone was ˜68 nm. By contrast, the hydrodynamic radius of CARBOPOL 971PNF™+Env polypeptide complex was ˜86 nm. This increased size of thecomplex indicated that the Env polypeptide was directly adsorbed on thecarbomer surface via charge-charge interaction. The Env polypeptide byitself has a hydrodynamic radius of >10 nm, so the CARBOPOL 971P NF™ wasclearly forming a complex with the Env polypeptide.

Example 4 Antigenic Integrity of Env Polypeptide Complexed withPolyanionic Carbomers

To verify that the polyanionic carbomers were not interfering with ordisrupting important conformational or neutralizing epitopes in the Envpolypeptide, the Env polypeptide-polyanionic carbomers (CARBOPOL 971PNF™) complexes were tested for their ability to bind receptor (CD4) anda monoclonal antibody (2G12, glycan-dependent). Soluble CD4 (sCD4) ormAb 2G12 were covalently immobilized on a CM5 sensor chip by aminecoupling. For the complexes, the Env polypeptide protein and CARBOPOL971P NF™ (final, 0.5%) were incubated at 4° C. for 1 hour. Afterincubation, 20-fold excess of HBS buffer was added and injected toanalyze binding to the immobilized ligands. 100 nM of Env polypeptide(gp140ΔV2), either alone or in complex with polyanionic carbomers wereinjected at 10 μl/min. As shown in FIG. 4, the Env polypeptide alonebound to sCD4 with an average RU (response unit) of ˜50. The Envpolypeptide complexes bound to sCD4 with ˜3-fold higher RU. Similarly,the Env polypeptide alone bound to mAb 2G12 with an RU of ˜150, whilethe Env polypeptide complexes bound with ˜3-fold higher RU. The Envpolypeptide both alone and complexed to polyanionic carbomers bound toboth ligands, indicating that the antigenic integrity of Env wasunaffected by the complex. The consistent 3-fold difference between theRU of the Env polypeptide alone and in complex was most likely be due tosize: the complex being larger in comparison to the Env polypeptidealone.

To further verify that the polyanionic carbomers were not interferingwith or disrupting important conformational or neutralizing epitopes inthe Env polypeptide, the Env polypeptide-polyanionic carbomers (CARBOPOL971P NF™) complexes were tested for their ability to bind receptor (CD4,here CD4-IgG2 is used as surrogate) and a monoclonal antibody (2G12,glycan-dependent) using capture ELISA. The capture ELISA was performedby coating MAXISORB™ plates with 2 μg/ml of D3724 mAb (in PBS) (100 μlper well), overnight at 4° C. The following day, the surface was blockedwith 1% BSA in PBS by incubating at 37° C. for one hour. The plates werethen washed three times (PBS+0.01% TWEEN 20™) and 1 μg/ml gp120 eitherpre-incubated with or without CARBOPOL 971P NF™, in 0.1% BSA+0.01%TRITON X-100™ (dilution buffer) was added to the plates. The plates wereincubated at room temperature (RT, 25° C.) for two hours. The plateswere washed three times and anti-CD4 IgG2, b12 or 17b, was added in aserial dilution starting at 1 μg/ml and then 2-fold diluted (in dilutionbuffer) thereafter. In cases where CD4i-induction using 17b mAb wasdesired, equimolar amount of soluble CD4 (sCD4) was added to gp120. Theplates were then incubated at RT for one hour and washed three times.Then anti-human HRP conjugated antibody was added to the reactions at1:10,000 (in dilution buffer). Following one hour incubation at RT, theplates were washed three times and developed using KPL's TMB substrates.All samples were evaluated in triplicate. A surface containing captureantibody, but no gp120 (but primary antibody, secondary antibody andsubstrate added to it), was used as control for each specific ligand.The optical density (OD) was determined using a microplate reader(Molecular Devices) at 450 nm. The results are shown in FIGS. 3(A)-(C).The Env proteins with and without CARBOPOL 971P NF™ bound the respectiveligands without any significant difference in binding affinity,indicating that the Env polypeptide do not denature or suffer antigenicalteration upon incubation in CARBOPOL 971P NF™ for up to 3 hours, whichis sufficient time to form complex before administration for vaccineevaluations. Taken together, these data indicate that the gp140 Envpolypeptide was stable in presence of CARBOPOL 971P NF™ preservingcritical conserved epitopes involved in receptor and co-receptorbinding.

Example 5 Immunogenicity of HIV-1 Subtype B Env Alone (Monovalent)Adjuvanted with CARBOPOL 971P NF™ (in Complex with the Env) or MF59™ orCARBOPOL 971P NF™ (in Complex with the Env)+MF59™ in a DNA Prime-ProteinBoost (IM) Regimen

This rabbit study is to compare CARBOPOL 971P NF™ (in complex) versusMF59™ versus CARBOPOL 971P NF™ (in complex)+MF59™ using a single(Subtype B SF162) gp140 Env polypeptide as immunogen. To confirm theimmunogenicity, rabbits were immunized with the subtype B SF162 Envpolypeptides in the complexes. New Zealand white rabbits, five pergroup, were immunized with 2 DNA primes (1 mg each immunization),followed by 25 μg of SF162 gp140ΔV2 (Env) protein boost with MF59™,CARBOPOL 971P NF™ or CARBOPOL 971P NF™+MF59™. This study was performedto compare the adjuvantation of CARBOPOL 971P NF™ versus MF59™ versusCARBOPOL 971P NF™+MF59™ using Subtype B SF162 gp140 Env polypeptide as(monovalent) immunogen. Four immunizations were administeredintramuscularly, in the gluteus, at weeks 0, 4, 12, and 24. The totalprotein dosage at each immunization was 25 μg. Serum samples werecollected prior to first immunization (pre-bleed) and at varioustime-points post each immunization (2wp2, 2wp3, 2wp4, 4wp4 and 15wp4bleed-out) and analyzed for binding and neutralization.

As measured using gp120-binding ELISA, Env polypeptide administered withMF59™ gave (geometric mean) titers of >10⁵ at two-weeks post-second(2wp2) and gave the highest titers of 10⁶ at 2wp3. The response to theEnv polypeptide administered with MF59™ did not improve post-fourthimmunization. The Env polypeptide complexed to CARBOPOL 971P NF™produced the highest titers of >10⁶ at 2wp3. Most significantly, Envpolypeptide complexed to CARBOPOL 971P NF™ and adjuvanted with MF59™gave the highest titers of all (about 10⁷ at 2wp3—see FIG. 5).

To further assess the immune response, the avidity of the gp140-specificserum antibodies produced by the vaccination protocol was assessed usingammonium thiocyanate ELISA (see FIG. 6). The avidity index provides anindication of the maturity of the antibodies produced.

The antibody avidity was similar following three or four immunizationsof Env in MF59™ or complexed to CARBOPOL 971P NF™, when sera wereevaluated 2-weeks post each immunization. In contrast, the antibodyavidity doubled following administration of Env polypeptide complexed toCARBOPOL 971P NF™ and adjuvanted with MF59™ at 2wp3 and 2wp4, incomparison to the two other regimens. This significant difference inimproving antibody avidity, including eliciting highly Env-specificbinding antibodies, using Env polypeptide complexed to CARBOPOL 971P NF™and adjuvanted with MF59™ is a noteworthy result and indicates that theCARBOPOL 971P NF™-Env polypeptide complexes and MF59™ work to furtherpotentiate binding antibody response, as observed in this case. Thiscould be due to synergistic effect where CARBOPOL™ works in delivery orcontrolled release of antigen and partial adjuvantation while MF59™works towards more potent immune-potentiation.

The ability of the Env-specific antibodies generated to neutralize adiverse panel of HIV-1 Env pseudoviruses based on Tiered categorization(See FIGS. 7A and B) was then tested. For most of the pseudovirusneutralization, the 2wp4 (p4) sera were more potent than that from 2wp3(p3). In comparison to Env administered with MF59™ or complexed withCARBOPOL 971P NF™ alone, Env complexed with CARBOPOL 971P NF™ andadjuvanted with MF59™ was most potent. However, no improvement inbreadth of the immune response was observed and only Tier 1A and 1Bviruses could be neutralized by the 2wp3/2wp4 vaccine sera (FIG. 7A)from all the comparing groups. MLV neutralization was performed ascontrol (FIG. 7B).

To further assess the ‘quality’ of humoral immune response elicitedpost-immunization, the specificity of antibody elicited in the rabbitsera was analyzed using a ‘serum mapping’ approach described by Y Li etal. (J Virol. 83(2): 1045-59, 2009). Using gp120 mutants (gp120ΔV1V2,gp120ΔV3, gp120D368R—CD4BS mutant, gp120I420R—a CD4i mutant) fordifferential adsorption of Env-specific antibodies, we found that amajority of the antibodies elicited using the gp140ΔV2 immunogens,either complexed to CARBOPOL 971P NF™, adjuvanted with MF59™, orcomplexed to CARBOPOL 971P NF™ and adjuvanted with MF59™, wereV3-specific. One rabbit in the group immunized with Env adjuvanted withMF59™ elicited CD4BS-antibodies. Other than this single animal, mostEnv-specific antibodies elicited were primarily directed to the gp120subunit, and more specifically to the V3-region of the glycoprotein.

In additional immunogenicity experiments, we observed that use ofcarbopol:Env complex, plus MF59™, with either monovalent (gp120/gp140)Env polypeptide or multivalent (gp120/gp140) Env polypeptide improvedthe neutralizing breadth and potency of the vaccine (rabbit) sera usingboth DNA primer-protein boost and protein only regimens. Thus, theimprovement in immunogenicity is not dependent upon the state (monomericor oligomeric) or valency (monovalent or multivalent) of the Envpolypeptide.

Example 6 Immunogenicity of HIV-1 Subtype C Env Derived from DifferentIsolates Alone (Monovalent) or in Combination (Multivalent) Formulatedwith CARBOPOL 971P NF™ in a DNA Prime-Protein Boost (IM) Regimen

This prime-boost study is to compare monovalent gp140 Env polypeptideadjuvanted with MF59™ to multivalent gp140 Env polypeptides adjuvantedwith MF59™. This study also compares multivalent gp140 Env polypeptidesadjuvanted with MF59™ versus multivalent gp140 Env polypeptidescomplexed to CARBOPOL™ and adjuvanted with MF59™. The immunogenicity ofHIV-1 subtype C gp140 Env derived from different isolates was evaluatedin a DNA Prime-Protein boost regimen. The Env polypeptide for the boostimmunizations were administered either as monovalent compositions(groups 1-7, and 10) or as multivalent compositions (groups 8 and 9)adjuvanted with MF59™. Group 8 animals were immunized with trivalentgp140 Env polypeptide adjuvanted with MF59™. In comparison, animals ingroup 9 were immunized with trivalent gp140 Env polypeptide complexedwith CARBOPOL 971P NF™ and adjuvanted with MF59 ™—so this is theCARBOPOL™+MF59 ™ group. For the multivalent/trivalent group, 50 μg(8.3+8.3+8.3 pgs of each Env polypeptide) of total Env polypeptide wasadministered.

TABLE 3 Immunization Study design of DNA prime-protein boost (IM) inrabbits of HIV-1 subtype C gp140 derived from different isolates andformulated with MF59 (TM) (for all monovalent group) and comparison ofmultivalent Env polypeptides with and without CARBOPOL 971P NF(TM) DNAPrime (weeks Protein Boost (weeks 12, Group 0, 4; dose - 1 mg) 24, 34;dose - 25 μg) 1 Du422.1 Du422.1 2 Du156.12 Du156.12 3 CAP45 CAP45 4ZM249M.PL1 ZM249M.PL1 5 HIV-25711-2 HIV-25711-2 6 CAP255 CAP255 7 CAP239CAP239  8* ZM249M.PL1 + CAP239 + ZM249M.PL1 + CAP239 + Du422.1 Du422.1 9* ZM249M.PL1 + CAP239 + ZM249M.PL1 + CAP239 + Du422.1 Du422.1 # 10 TV1 TV1 5 rabbits/group; IM immunizations (DNA and protein) DNA prime: 1mg/dose at weeks 0 and 4 Protein boost: 25 μg with MF59/dose at weeks12, 24, and 34 *Equal composition of each Env in DNA prime and proteinboost # Protein boost adjuvanted with MF59(TM) + CARBOPOL 971P NF(TM)

Neutralization breadth after vaccination with HIV-1 subtype C gp140 Envpolypeptide formulated in MF59™ only, i.e., without CARBOPOL 971P NF™(except for group 9) in Rabbits for Tier 1a and Tier 1b as well as forTier 2 (pseudo-) viruses using sera collected at two weeks post fourth(2wp4) immunization are shown in FIGS. 8A and B. In particular, FIG. 8shows the results as a heat map showing breadth and potency (in ID50titers) of serum neutralization of HIV-1 pseudoviruses. The breadth andpotency of serum neutralization of HIV-1 pseudoviruses was assessed asfollows. Sera were analyzed 2 weeks post 4th immunization. Sera fromeach rabbit within groups were tested against the tiered (Tier 1a, Tier1b and Tier 2) virus panel of SF162, MN.3, Bal.26, Du156.12, Du422.1,ZM249M.PL1, MW965.26, TV1c21 and CAP239 in a single-cycle TZM-b1pseudovirus assay. Neutralization was assessed using molecularly clonedpseudoviruses and a luciferase reporter gene assay in TZM-b1 cells.Briefly, a total of 200 TCID50 pseudovirus/well were added to dilutedsera samples and incubated at 37° C. for 1 hour. Following incubation,10,000 cells/well in DEAE-dextran-containing media were added andincubated for 48 hrs at 37° C. The final concentration of DEAE-dextranwas 10 μg/ml. Single round of infection HIV-1 Env pseudoviruses wereprepared by co-transfection of 293T cells with an envelope expressionplasmid containing a full-length gp160 env gene along with anenv-deficient HIV-1 backbone vector (pSG3Δenv), using TransIT®-LT1transfection reagent (Mirus Bio Corp., Madison, Wis.). After 48 hrs, thecell culture supernatant containing the pseudovirus was filtered througha 0.45 μm filter. Neutralizing activity was measured as reductions inluciferase gene expression. The percent reduction in relativeluminescence units (RLU) was calculated relative to the RLU in thepresence of pre-immunization serum. Neutralizing antibody titers againstSF162 strain were determined using 3-fold serially diluted sera samples.The breadth of neutralizing antibodies in sera was assessed at a serumdilution of 1:20. The percent neutralization was corrected fornon-specific inhibition using the formula described previously with MLVas a control virus.

Potent neutralization of Subtype C Tier 1a MW965.26 pseudovirus,appreciable neutralization of Subtype B Tier 1a pseudoviruses, and poorneutralization of Tier 1b pseudoviruses was observed. Themultivalent/trivalent arm (group 8) showed no distinct advantage inneutralizing ID50 titer over single envelope antigens however theCARBOPOL 971P NF™+MF59™ adjuvant arm (group 9) showed enhanced potency.

Neutralization ID50 titers of Tier 1 isolates (2wp3 (p3), 2wp4 (p4), &2wp5 (p5)): The fifth immunization did not improve titers in most casesas shown in FIGS. 9A, B (Tier 1a) and C (Tier 1b) or in no cases forTier 2, respectively (FIGS. 9D and E).

FIG. 10 shows total antibody-binding titers against TV1 gp140 Envpolypeptide as measured by gp120-binding ELISA. The background titer forthe prebleeds (as control) is also included. The antibody titers weredetermined by ELISA using TV1 gp140 Env polypeptide as the coatingprotein. The data values shown represent geometric mean titers (GMT) offive rabbits individually assayed in triplicates per group. All antigenselicited robust antibody geometric mean titers (GMT), with peak GMT forall antigens exceeding 10⁶.

The antibody avidity was evaluated for sera collected from all groups(FIG. 11). Avidity was determined by NH₄SCN displacement ELISA usingTV1c8.2 rgp140-o as the coating antigen as described by I. K. Srivastavaet al. (J. Virol. 2002).

Example 7 Immunogenicity of CARBOPOL 971P NF™:gp140 Env PolypeptideComplexes in Rabbits in Protein Only (IM) Regimen

This study, in contrast to studies in Examples 5 and 6, is a proteinonly study. Examples 5 and 6 show that in both monovalent andmultivalent Env polypeptide immunizations in DNA prime-protein boostregimen that CARBOPOL 971P NF™+MF59™ was more effective. This studydemonstrates that CARBOPOL 971P NF™+MF59™ was equally effective inprotein-only regimen and there is no difference in multivalentimmunizations when either co-administered or given sequentially.

Immunization of rabbits with HIV-1 subtype C gp140 Env polypeptideformulated with CARBOPOL 971P NF™+MF59™ (see Table 4). 25 μg of eachindividual gp140 Env polypeptide from the isolates listed in Table 4(all groups except 8) was administered per rabbit. For group 8, 6.25 μgof gp140 Env polypeptide from each strain was combined to give a finaldose of 25 μg gp140 Env polypeptide. For each group, five New ZealandWhite rabbits were used in this immunogenicity study. Env polypeptidewere administered in complex with CARBOPOL 971P NF™ adjuvanted withMF59™. Serum samples were collected prior to first immunization(pre-bleed) and two weeks following each immunization.

TABLE 4 Immunization study design of HIV-1 subtype C gp140Env formulatedwith CARBOPOL 971P NF(TM) + MF59(TM) in Rabbits Group Protein Only(weeks 0. 4, 12, 24; dose - 25 μg) 1 Du156.12 gp140 2 Du422.1 gp140 3ZM249M.PL1 gp140 4 CAP239 gp140 5 TV1 gp140 6 TV1 gp140 ΔV2 7 SF162gp140 ΔV2  8* ZM249M.PL1 + CAP239 + Du422.1 + TV1 gp140  9# CAP239gp140/Du422.1 gp140/ZM249M.PL1 gp140/TV1 gp140 Protein: 25 μg with MF59and CARBOPOL 971P NF(TM)/dose at weeks 0, 4, 12 and 24 *Equalcomposition of each Env polypeptide (6.25 μg each) #Sequentialimmunization: 25 μg single Env polypeptide immunization 5 rabbits/group;IM immunizations (protein only)

The neutralization breadth (in ID50 titers) was determined aftervaccination with HIV-1 subtype C gp140Env formulated with CARBOPOL 971PNF™+MF59™ for all groups with sera collected at 2wp3 (see FIGS. 12A andB). Sera were tested against the HIV-1 subtype C Tier 1a, b and Tier 2pseudovirus panels in a single-cycle TMZ-b1 pseudovirus assay, asdescribed above. As shown in FIG. 12, 2wp3 sera readily neutralized Tier1a viruses, but mostly failed to neutralize Tier 1b or Tier 2 viruses.Multivalent or sequential immunization of gp140 Env polypeptides did notimprove the overall immune response.

The neutralization breadth (in ID50 titers) was determined aftervaccination with HIV-1 subtype C gp140 Env polypeptide formulated withCARBOPOL 971P NF™+MF59™ for all groups with sera collected at 2wp4 (seeFIGS. 13A, B and C). Sera were tested against an extended HIV-1 subtypeB and C virus panel in a single-cycle TMZ-b1 pseudovirus assay, asdescribed above. As observed, at 2wp4, serum was more potent andneutralized majority of the Tier 1a and Tier 2a viruses (although withlower ID50 titers). Some low neutralization of Tier 2 viruses was alsoobserved. Overall, 2wp4 sera provided better neutralization than 2wp3sera (compare FIGS. 12 and 13), emphasizing the need for a secondaryprotein boost.

Potent neutralization of Tier 1 isolates post 3^(rd) and 4^(th)immunization (Tier 1a: FIGS. 14A-B; Tier 1b: FIG. 14C). The fourthimmunization increased titers against Tier 1b TV1.21 virus. Tier 2:FIGS. 14D-E in a single-cycle TMZ-b1 pseudovirus assay, as describedabove.

Evaluation of total antibody titers was performed by ELISA using TV1gp140 Env polypeptide as the coating antigen as described by I. K.Srivastava et al. (J. Virol. 2002). (FIG. 15—Group 8 (multivalent):ZM249M.PL1+CAP239+Du422.1+TV1 gp140; Group 9 (sequential): CAP239gp140/Du422.1 gp140/ZM249M.PL1 gp140/TV1 gp140).

The avidity of the antibodies was determined as described above. (FIG.16—Group 8 (multivalent): ZM249M.PL1+CAP239+Du422.1+TV1 gp140; Group 9(sequential): CAP239 gp140/Du422.1 gp140/ZM249M.PL1 gp140/TV1 gp140).

Example 8 Evaluate Effect of DNA Prime-Protein Boost Versus Protein OnlyImmunizations with CARBOPOL 971P NF™:Env Complexes Adjuvanted with MF59™

To confirm whether the improvement in the immunogenicity required a DNAprime, data from the previously shown immunization experiments using 2DNA-prime followed by 3 protein-boost (see Table 3) or 4 protein boosts(see Table 4) immunizations of the CARBOPOL 971P NF™:Env complexesadjuvanted with MF59™ were further analyzed. A number of different gp140Env polypeptides generated from subtype C isolates were tested. Allgp140 isolates tested showed an improvement in the immunogenicity (SeeTable 5—(2wp2—2-weeks after second protein boost and after two DNAprimes; 2wp4—2-weeks after fourth protein boost but no DNA prime). ≧60%of the animals exhibited >90% neutralization potency against a Subtype Cpseudovirus, MW965.1. The priming via DNA or other vector could bebeneficial for eliciting key immune response such as T-cell response(not measured here). However, from just the antibody-response, theeffect of improved neutralization is not dependent upon DNA priming.Further, the improved immunogenicity is not limited to the SF162 isolateor even Subtype B isolates.

Significantly, when comparing average viral inhibition of pseudoviruses,between MF59™ without CARBOPOL™) and MF59™ with CARBOPOL 971P NF™, weobserved that in each case MF59™ with CARBOPOL 971P NF™ generated betterfunctional response than MF59™ only group.

TABLE 5 Neutralization Potency of Env polypeptides adjuvanted with MF59(TM) against Subtype C isolates No CARBOPOL(TM) CARBOPOL 971P NF(TM)Sera >90% Average Sera >90% Average Isolates inhibition. inhibitioninhibition inhibition (Gp140) (2wp4) (%) (2wp2) (%) Du156.12 1/5 72 4/592 Du422.1 1/5 76 4/5 88 ZM249.PL1 1/5 81 3/5 90 CAP239 1/5 79 3/5 90TV1 1/5 85 3/5 89 2/5 (ΔV2) 88 ZM249.PL1 + 1/5 81 4/5 92 CAP239 +Du422.1

Example 9 Immunogenicity of CARBOPOL 971P NF™:Env gp120 PolypeptideComplexes Adjuvanted with MF59™ in Rabbits

As opposed to other studies described above, where predominantly gp140Env polypeptides were used, here we use gp120 Env polypeptide toevaluate if the improved response, when using CARBOPOL 971P NF™+MF59™,was broadly applicable to all Env constructs regardless ofoligomerization state, size, etc. Immunization of rabbits with HIV-1subtype C gp120 Env polypeptide formulated with CARBOPOL 971P NF™ andMF59™ (see Table 6). For each group shown in Table 6, five New ZealandWhite rabbits were used in the immunogenicity study. Rabbits wereimmunized with 25 μg of gp120 protein formulated in Carbopol™ and MF59.For the final group, group 11, gp120 proteins from four differentstrains were combined, 6.25 μg each, totaling 25 μg of gp120 protein perdose. Protein only vaccinations were administered on weeks 0, 4, 12 and24. Serum samples were collected prior to immunization (pre-bleed) and 2weeks following 2nd (2wp2), 3rd (2wp3) and 4th (2wp4) immunization.Final serum was collected 4 weeks after final immunization (4wp4).

TABLE 6 Immunization study design of HIV-1 subtype C gp120 Envpolypeptide adjuvanted with CARBOPOL 971P NF(TM) + MF59(TM) in a proteinonly study (IM) in Rabbits Group Protein Only (weeks 0, 4, 12, 24;dose - 25 μg) 1 Du156.12 gp120 2 Du422.1 gp120 3 ZM249M.PL1 gp120 4CAP45 gp120 5 CAP84 gp120 6 CAP239 gp120 7 TV1 gp120 8 SF162 gp120 9 TV1gp140 10  SF162 gp140 11# 1. CAP239; 2. Du422.1; 3. ZM249; 4. TV1 (allgp120) 5 rabbits/group; IM immunizations Protein: 25 μg with MF59(TM)and CARBOPOL 971P NF(TM)/dose at weeks 0, 4, 12 and 24 #Sequentialimmunization: 25 μg single Env/immunization

Immunization in Rabbits with Subtype C CARBOPOL 971P NF™:Env gp120complexes adjuvanted with MF59™. Neutralization breadth (ID50 titers)determined with sera collected at 2wp3 (FIGS. 17A-F) in a single-cycleTMZ-b1 pseudovirus assay, as described above.

Immunization in Rabbits with Subtype C CARBOPOL 971P NF™:Env gp120complexes adjuvanted with MF59™. Neutralization (ID50 titers) wasdetermined with sera collected at 2wp3 against Tier 1a and Tier 2 HIV-1subtype C pseudovirus panels (FIGS. 18A-B) in a single-cycle TMZ-b1pseudovirus assay, as described above.

As described above, mAb competition ELISA was conducted againstimmobilized TV1 gp140 Env polypeptide with pooled sera (1:100 dilution)collected 2 weeks post 4^(th) immunization with subtype C gp120 (week22) (FIG. 19), to dissect antibody specificity against Env.

Example 10 Immunogenicity of CARBOPOL 971P NF™:Env gp120 PolypeptideComplexes Adjuvanted with MF59™ in Protein Only (IM) Study in GuineaPigs

Immunization of Guinea pigs with HIV-1 subtype C gp120 Env polypeptideformulated with CARBOPOL 971P NF™+MF59™ (see Table 7): Guinea-pigs wereimmunized with 25 μg of gp120 protein formulated in Carbopol™ and MF59™.Protein only vaccinations were administered on weeks 0, 4, 12 and 24.Serum samples were collected prior to immunization (pre-bleed) and 2weeks following 2^(nd) (2wp2), 3^(rd) (2wp3) and 4^(th) (2wp4)immunization. Final sera were collected 4 weeks after final immunization(4wp4).

TABLE 7 Immunization schedule of HIV-1 subtype C gp120 Env formulatedwith CARBOPOL 971P NF(TM) in Guinea pigs Group Protein Only (weeks 0, 4,12, 24; dose - 25 μg) 1 Du156.12 gp120 2 Du422.1 gp120 3 ZM249M.PL1gp120 4 CAP45 gp120 5 CAP84 gp120 6 CAP239 gp120 7 TV1 gp120 8 SF162gp120 9 TV1 gp140 10 SF162 gp140 5 Guinea pigs/group; IM immunizationsProtein: 25 μg with MF59(TM) and CARBOPOL 971P NF(TM)/dose at weeks 0,4, 12 and 24

Neutralization breadth (ID50 titers) was determined with sera collectedat 2wp3 (FIGS. 20A-F) in a single-cycle TMZ-b1 pseudovirus assay, asdescribed above. Neutralization (ID50 titers) was determined with seracollected at 2wp3 against Tier 1a and Tier 2 HIV-1 subtype C viruspanels (FIG. 21) in a single-cycle TMZ-b1 pseudovirus assay, asdescribed above.

As described above, mAb competition ELISA was conducted againstimmobilized TV1 gp140 Env polypeptide with pooled sera (1:500 dilution)collected 2 weeks post 3^(rd) (FIG. 22; week 14) or 2 weeks post 4^(th)(FIG. 23; week 26) immunization with subtype C gp120), to assessantibody specificity against Env.

Example 11 Evaluate Adverse Reactions with CARBOPOL 971™:Env ComplexesAdjuvanted with MF59™ after Injection in Rabbits

Rabbits were observed for overall reactogenicity and for any obvioushealth problems against CARBOPOL 971™ after immunization (See Table 8).MF59™ has been used in multiple species, including humans, and found tobe safe. Therefore, the goal was to determine if CARBOPOL 971P NF™ incombination with MF59™ causes any adverse reactivity. Since MF59™ doesnot cause any such reactivity, any observed reactogenicity would likelybe due to CARBOPOL 971P NF™. However, no immediate local reactogenicitypost injection was observed at the injection site. Development of smalledema and erythema was detected within 1-2 hours following eachinjection, which disappeared after 24 hours. As shown in FIGS. 24A-K, nosignificant loss of body weight occurred immediately after the firstvaccination and all animals from the three groups shown in FIGS. 24A-Kcontinued to gain weight for more than 140 days during the course of thestudy. Rabbits were monitored for body-weight one day beforeimmunization, 24, 48 and 72 hours post-vaccination (see FIG. 24). Localreactivity and obvious health problems were monitored at 24, 48 and 72hours post-vaccination. All observations were recorded in alog-notebook. Overall, no obvious health problems (NOHP) were observedin rabbits vaccinated with CARBOPOL 971™:Env polypeptide adjuvanted withMF59™; also no significant loss of body-weight post-immunization wasobserved. This indicates that administration of CARBOPOL 971™ was safein rabbits under the present settings.

TABLE 8 Rabbit study: Animals observed for loss of body- weight and anyobvious health problems during or after immunization of gp120 withCarbopol and MF59 Group Env Protein Animals 1 Du156.12 gp120 1-5 2Du422.1 gp120  6-10 3 ZM249M.PL1 gp120 11-15 4 CAP45 gp120 16-20 5 CAP84gp120 21-25 6 CAP239 gp120 26-30 7 TV1 gp120 31-35 8 SF162 gp120 36-40 9TV1 gp140 41-45 10  SF162 gp140 46-50 11# 1. CAP239; 2. Du422.1; 3.51-55 ZM249; 4. TV1 (all gp120)

<210> SEQ ID NO 1 <211> LENGTH: 1419 <212> TYPE: DNA <213>ORGANISM: Human immunodeficiency virus <400> SEQUENCE: 1gtagaaaaat tgtgggtcac agtctattat ggggtacctg tgtggaaaga agcaaccacc 60actctatttt gtgcatcaga tgctaaagcc tatgacacag aggtacataa tgtctgggcc 120acacatgcct gtgtacccac agaccctaac ccacaagaaa tagtattgga aaatgtgaca 180gaaaatttta acatgtggaa aaataacatg gtagaacaga tgcatgagga tataatcagt 240ttatgggatc aaagtctaaa gccatgtgta aagttaaccc cactctgtgt tactctacat 300tgcactaatt tgaagaatgc tactaatacc aagagtagta attggaaaga gatggacaga 360ggagaaataa aaaattgctc tttcaaggtc accacaagca taagaaataa gatgcagaaa 420gaatatgcac ttttttataa acttgatgta gtaccaatag ataatgataa tacaagctat 480aaattgataa attgtaacac ctcagtcatt acacaggcct gtccaaaggt atcctttgaa 540ccaattccca tacattattg tgccccggct ggttttgcga ttctaaagtg taatgataag 600aagttcaatg gatcaggacc atgtacaaat gtcagcacag tacaatgtac acatggaatt 660aggccagtag tgtcaactca attgctgtta aatggcagtc tagcagaaga aggggtagta 720attagatctg aaaatttcac agacaatgct aaaactataa tagtacagct gaaggaatct 780gtagaaatta attgtacaag acctaacaat aatacaagaa aaagtataac tataggaccg 840gggagagcat tttatgcaac aggagacata ataggagata taagacaagc acattgtaac 900attagtggag aaaaatggaa taacacttta aaacagatag ttacaaaatt acaagcacaa 960tttgggaata aaacaatagt ctttaagcaa tcctcaggag gggacccaga aattgtaatg 1020cacagtttta attgtggagg ggaatttttc tactgtaatt caacacagct ttttaatagt 1080acttggaata atactatagg gccaaataac actaatggaa ctatcacact cccatgcaga 1140ataaaacaaa ttataaacag gtggcaggaa gtaggaaaag caatgtatgc ccctcccatc 1200agaggacaaa ttagatgctc atcaaatatt acaggactgc tattaacaag agatggtggt 1260aaagagatca gtaacaccac cgagatcttc agacctggag gtggagatat gagggacaat 1320tggagaagtg aattatataa atataaagta gtaaaaattg agccattagg agtagcaccc 1380accaaggcaa agagaagagt ggtgcagaga gaaaaaaga 1419 <210> SEQ ID NO 2 <211>LENGTH: 1932 <212> TYPE: DNA <213>ORGANISM: Human immunodeficiency virus <400> SEQUENCE: 2gtagaaaaat tgtgggtcac agtctattat ggggtacctg tgtggaaaga agcaaccacc 60actctatttt gtgcatcaga tgctaaagcc tatgacacag aggtacataa tgtctgggcc 120acacatgcct gtgtacccac agaccctaac ccacaagaaa tagtattgga aaatgtgaca 180gaaaatttta acatgtggaa aaataacatg gtagaacaga tgcatgagga tataatcagt 240ttatgggatc aaagtctaaa gccatgtgta aagttaaccc cactctgtgt tactctacat 300tgcactaatt tgaagaatgc tactaatacc aagagtagta attggaaaga gatggacaga 360ggagaaataa aaaattgctc tttcaaggtc accacaagca taagaaataa gatgcagaaa 420gaatatgcac ttttttataa acttgatgta gtaccaatag ataatgataa tacaagctat 480aaattgataa attgtaacac ctcagtcatt acacaggcct gtccaaaggt atcctttgaa 540ccaattccca tacattattg tgccccggct ggttttgcga ttctaaagtg taatgataag 600aagttcaatg gatcaggacc atgtacaaat gtcagcacag tacaatgtac acatggaatt 660aggccagtag tgtcaactca attgctgtta aatggcagtc tagcagaaga aggggtagta 720attagatctg aaaatttcac agacaatgct aaaactataa tagtacagct gaaggaatct 780gtagaaatta attgtacaag acctaacaat aatacaagaa aaagtataac tataggaccg 840gggagagcat tttatgcaac aggagacata ataggagata taagacaagc acattgtaac 900attagtggag aaaaatggaa taacacttta aaacagatag ttacaaaatt acaagcacaa 960tttgggaata aaacaatagt ctttaagcaa tcctcaggag gggacccaga aattgtaatg 1020cacagtttta attgtggagg ggaatttttc tactgtaatt caacacagct ttttaatagt 1080acttggaata atactatagg gccaaataac actaatggaa ctatcacact cccatgcaga 1140ataaaacaaa ttataaacag gtggcaggaa gtaggaaaag caatgtatgc ccctcccatc 1200agaggacaaa ttagatgctc atcaaatatt acaggactgc tattaacaag agatggtggt 1260aaagagatca gtaacaccac cgagatcttc agacctggag gtggagatat gagggacaat 1320tggagaagtg aattatataa atataaagta gtaaaaattg agccattagg agtagcaccc 1380accaaggcaa agagaagagt ggtgcagaga gaaaaaagag cagtgacgct aggagctatg 1440ttccttgggt tcttgggagc agcaggaagc actatgggcg cacggtcact gacgctgacg 1500gtacaggcca gacaattatt gtctggtata gtgcaacagc agaacaattt gctgagagct 1560attgaggcgc aacagcatct gttgcaactc acagtctggg gcatcaagca gctccaggca 1620agagtcctgg ctgtggaaag atacctaaag gatcaacagc tcctagggat ttggggttgc 1680tctggaaaac tcatttgcac cactgctgtg ccttggaatg ctagttggag taataaatct 1740ctggatcaga tttggaataa catgacctgg atggagtggg agagagaaat tgacaattac 1800acaaacttaa tatacacctt aattgaagaa tcgcagaacc aacaagaaaa gaatgaacaa 1860gaattattag aattggataa gtgggcaagt ttgtggaatt ggtttgacat atcaaaatgg 1920ctgtggtata ta 1932 <210> SEQ ID NO 3 <211> LENGTH: 2457 <212> TYPE: DNA<213> ORGANISM: Human immunodeficiency virus <400> SEQUENCE: 3gtagaaaaat tgtgggtcac agtctattatg gggtacctgt gtggaaaga agcaaccacc 60actctatttt gtgcatcaga tgctaaagcct atgacacaga ggtacataa tgtctgggcc 120acacatgcct gtgtacccac agaccctaacc cacaagaaat agtattgga aaatgtgaca 180gaaaatttta acatgtggaa aaataacatgg tagaacagat gcatgagga tataatcagt 240ttatgggatc aaagtctaaa gccatgtgtaa agttaacccc actctgtgt tactctacat 300tgcactaatt tgaagaatgc tactaatacca agagtagtaa ttggaaaga gatggacaga 360ggagaaataa aaaattgctc tttcaaggtca ccacaagcat aagaaataa gatgcagaaa 420gaatatgcac ttttttataa acttgatgtag taccaataga taatgataa tacaagctat 480aaattgataa attgtaacac ctcagtcatta cacaggcctg tccaaaggt atcctttgaa 540ccaattccca tacattattg tgccccggctg gttttgcgat tctaaagtg taatgataag 600aagttcaatg gatcaggacc atgtacaaatg tcagcacagt acaatgtac acatggaatt 660aggccagtag tgtcaactca attgctgttaa atggcagtct agcagaaga aggggtagta 720attagatctg aaaatttcac agacaatgcta aaactataat agtacagct gaaggaatct 780gtagaaatta attgtacaag acctaacaata atacaagaaa aagtataac tataggaccg 840gggagagcat tttatgcaac aggagacataa taggagatat aagacaagc acattgtaac 900attagtggag aaaaatggaa taacactttaa aacagatagt tacaaaatt acaagcacaa 960tttgggaata aaacaatagt ctttaagcaat cctcaggagg ggacccaga aattgtaatg 1020cacagtttta attgtggagg ggaatttttct actgtaattc aacacagct ttttaatagt 1080acttggaata atactatagg gccaaataaca ctaatggaac tatcacact cccatgcaga 1140ataaaacaaa ttataaacag gtggcaggaag taggaaaagc aatgtatgc ccctcccatc 1200agaggacaaa ttagatgctc atcaaatatta caggactgct attaacaag agatggtggt 1260aaagagatca gtaacaccac cgagatcttca gacctggagg tggagatat gagggacaat 1320tggagaagtg aattatataa atataaagtag taaaaattga gccattagg agtagcaccc 1380accaaggcaa agagaagagt ggtgcagagag aaaaaagagc agtgacgct aggagctatg 1440ttccttgggt tcttgggagc agcaggaagca ctatgggcgc acggtcact gacgctgacg 1500gtacaggcca gacaattatt gtctggtatag tgcaacagca gaacaattt gctgagagct 1560attgaggcgc aacagcatct gttgcaactca cagtctgggg catcaagca gctccaggca 1620agagtcctgg ctgtggaaag atacctaaagg atcaacagct cctagggat ttggggttgc 1680tctggaaaac tcatttgcac cactgctgtgc cttggaatgc tagttggag taataaatct 1740ctggatcaga tttggaataa catgacctgga tggagtggga gagagaaat tgacaattac 1800acaaacttaa tatacacctt aattgaagaat cgcagaacca acaagaaaa gaatgaacaa 1860gaattattag aattggataa gtgggcaagtt tgtggaattg gtttgacat atcaaaatgg 1920ctgtggtata taaaaatatt cataatgatag taggaggttt agtaggttt aaggatagtt 1980tttactgtgc tttctatagt gaatagagtta ggcagggata ctcaccatt atcatttcag 2040acccgcttcc cagccccaag gggacccgaca ggcccgaagg aatcgaaga agaaggtgga 2100gagagagaca gagacagatc cagtccattag tgcatggatt attagcact catctgggac 2160gatctacgga gcctgtgcct cttcagctacc accgcttgag agacttaat cttgattgca 2220gcgaggattg tggaacttct gggacgcaggg ggtgggaagc cctcaagta ttgggggaat 2280ctcctgcagt attggattca ggaactaaaga atagtgctgt tagtttgtt tgatgccata 2340gctatagcag tagctgaggg gacagatagga ttatagaagt agcacaaag aattggtaga 2400gcttttctcc acatacctag aagaataagac agggctttga aagggcttt gctataa 2457<210> SEQ ID NO 4 <211> LENGTH: 1453 <212> TYPE: DNA <213>ORGANISM: Artificial Sequence <220> FEATURE: <223>OTHER INFORMATION: Description ofArtificialSequence: gp120.modSF162<400> SEQUENCE: 4gaattcgcca ccatggatgc aatgaagaga gggctctgct gtgtgctgct gctgtgtgga 60gcagtcttcg tttcgcccag cgccgtggag aagctgtggg tgaccgtgta ctacggcgtg 120cccgtgtgga aggaggccac caccaccctg ttctgcgcca gcgacgccaa ggcctacgac 180accgaggtgc acaacgtgtg ggccacccac gcctgcgtgc ccaccgaccc caacccccag 240gagatcgtgc tggagaacgt gaccgagaac ttcaacatgt ggaagaacaa catggtggag 300cagatgcacg aggacatcat cagcctgtgg gaccagagcc tgaagccctg cgtgaagctg 360acccccctgt gcgtgaccct gcactgcacc aacctgaaga acgccaccaa caccaagagc 420agcaactgga aggagatgga ccgcggcgag atcaagaact gcagcttcaa ggtgaccacc 480agcatccgca acaagatgca gaaggagtac gccctgttct acaagctgga cgtggtgccc 540atcgacaacg acaacaccag ctacaagctg atcaactgca acaccagcgt gatcacccag 600gcctgcccca aggtgagctt cgagcccatc cccatccact actgcgcccc cgccggcttc 660gccatcctga agtgcaacga caagaagttc aacggcagcg gcccctgcac caacgtgagc 720accgtgcagt gcacccacgg catccgcccc gtggtgagca cccagctgct gctgaacggc 780agcctggccg aggagggcgt ggtgatccgc agcgagaact tcaccgacaa cgccaagacc 840atcatcgtgc agctgaagga gagcgtggag atcaactgca cccgccccaa caacaacacc 900cgcaagagca tcaccatcgg ccccggccgc gccttctacg ccaccggcga catcatcggc 960gacatccgcc aggcccactg caacatcagc ggcgagaagt ggaacaacac cctgaagcag 1020atcgtgacca agctgcaggc ccagttcggc aacaagacca tcgtgttcaa gcagagcagc 1080ggcggcgacc ccgagatcgt gatgcacagc ttcaactgcg gcggcgagtt cttctactgc 1140aacagcaccc agctgttcaa cagcacctgg aacaacacca tcggccccaa caacaccaac 1200ggcaccatca ccctgccctg ccgcatcaag cagatcatca accgctggca ggaggtgggc 1260aaggccatgt ecgccccccc catccgcggc cagatccgct gcagcagcaa catcaccggc 1320ctgctgctga cccgcgacgg cggcaaggag ateogcaaea ccaccgagat cttccgccce 1380ggcggcggcg acatgcgcga caactggcgc agcgagctgt acaagtacaa ggtggcgaag 1440atcgagcccc tgg 1453 <210> SEQ ID NO 5 <211> LENGTH: 1387 <212> TYPE: DNA<213> ORGANISM: Artificial Sequence <220> FEATURE: <223>OTHER INFORMATION: Description of Artificial Sequence:gp120.modSF162.delV2 <400> SEQUENCE: 5gaattcgcca ccatggatgc aatgaagaga gggctctgct gtgtgctgct gctgtgtgga 60gcagtcttcg tttcgcccag cgccgtggag aagctgtggg tgaccgtgta ctacggcgtg 120cccgtgtgga aggaggccac caccaccctg ttctgcgcca gcgacgccaa ggcctacgac 180accgaggtgc acaacgtgtg ggccacccac gcctgcgtgc ccaccgaccc caacccccag 240gagatcgtgc tggagaacgt gaccgagaac ttcaacatgt ggaagaacaa catggtggag 300cagatgcacg aggacatcat cagcctgtgg gaccagagcc tgaagccctg cgtgaagctg 360acccccctgt gcgtgaccct gcactgcacc aacctgaaga acgccaccaa caccaagagc 420agcaactgga aggagatgga ccgcggcgag atcaagaact gcagcttcaa ggtgggcgcc 480ggcaagctga tcaactgcaa caccagcgtg atcacccagg cctgccccaa ggtgagcttc 540gagcccatcc ccatccacta ctgcgccccc gccggcttcg ccatcctgaa gtgcaacgac 600aagaagttca acggcagcgg cccctgcacc aacgtgagca ccgtgcagtg cacccacggc 660atccgccccg tggtgagcac ccagctgctg ctgaacggca gcctggccga ggagggcgtg 720gtgatccgca gcgagaactt caccgacaac gccaagacca tcatcgtgca gctgaaggag 780agcgtggaga tcaactgcac ccgccccaac aacaacaccc gcaagagcat caccatcggc 840cccggccgcg ccttctacgc caccggcgac atcatcggcg acatccgcca ggcccactgc 900aacatcagcg gcgagaagtg gaacaacacc ctgaagcaga tcgtgaccaa gctgcaggcc 960cagttcggca acaagaccat cgtgttcaag cagagcagcg gcggcgaccc cgagatcgtg 1020atgcacagct tcaactgcgg cggcgagttc ttctactgca acagcaccca gctgttcaac 1080agcacctgga acaacaccat cggccccaac aacaccaacg gcaccatcac cctgccctgc 1140cgcatcaagc agatcatcaa ccgctggcag gaggtgggca aggccatgta cgcccccccc 1200atccgcggcc agatccgctg cagcagcaac atcaccggcc tgctgctgac ccgcgacggc 1260ggcaaggaga tcagcaacac caccgagatc ttccgccccg gcggcggcga catgcgcgac 1320aactggcgca gcgagctgta caagtacaag gtggtgaaga tcgagcccct gggcgtggcc 1380cccacca 1387 <210> SEQ ID NO 6 <211> LENGTH: 1323 <212> TYPE: DNA <213>ORGANISM: Artificial Sequence <220> FEATURE: <223>OTHER INFORMATION: Description of Artificial Sequence:gp120.modSF162.delVlV2 <400> SEQUENCE: 6gaattcgcca ccatggatgc aatgaagaga gggctctgct gtgtgctgct gctgtgtgga 60gcagtcttcg tttcgcccag cgccgtggag aagctgtggg tgaccgtgta ctacggcgtg 120cccgtgtgga aggaggccac caccaccctg ttctgcgcca gcgacgccaa ggcctacgac 180accgaggtgc acaacgtgtg ggccacccac gcctgcgtgc ccaccgaccc caacccccag 240gagatcgtgc tggagaacgt gaccgagaac ttcaacatgt ggaagaacaa catggtggag 300cagatgcacg aggacatcat cagcctgtgg gaccagagcc tgaagccctg cgtgaagctg 360acccccctgt gcgtgggcgc cggcaactgc cagaccagcg tgatcaccca ggcctgcccc 420aaggtgagct tcgagcccat ccccatccac tactgcgccc ccgccggctt cgccatcctg 480aagtgcaacg acaagaagtt caacggcagc ggcccctgca ccaacgtgag caccgtgcag 540tgcacccacg gcatccgccc cgtggtgagc acccagctgc tgctgaacgg cagcctggcc 600gaggagggcg tggtgatccg cagcgagaac ttcaccgaca acgccaagac catcatcgtg 660cagctgaagg agagcgtgga gatcaactgc acccgcccca acaacaacac ccgcaagagc 720atcaccatcg gccccggccg cgccttctac gccaccggcg acatcatcgg cgacatccgc 780caggcccact gcaacatcag cggcgagaag tggaacaaca ccctgaagca gatcgtgacc 840aagctgcagg cccagttcgg caacaagacc atcgtgttca agcagagcag cggcggcgac 900cccgagatcg tgatgcacag cttcaactgc ggcggcgagt tcttctactg caacagcacc 960cagctgttca acagcacctg gaacaacacc atcggcccca acaacaccaa cggcaccatc 1020accctgccct gccgcatcaa gcagatcatc aaccgctggc aggaggtggg caaggccatg 1080tacgcccccc ccatccgcgg ccagatccgc tgcagcagca acatcaccgg cctgctgctg 1140acccgcgacg gcggcaagga gatcagcaac accaccgaga tcttccgccc cggcggcggc 1200gacatgcgcg acaactggcg cagcgagctg tacaagtaca aggtggtgaa gatcgagccc 1260ctgggcgtgg cccccaccaa ggccaagcgc cgcgtggtgc agcgcgagaa gcgctaactc 1320gag 1323 <210> SEQ ID NO 7 <211> LENGTH: 2025 <212> TYPE: DNA <213>ORGANISM: Artificial Sequence <220> FEATURE: <223>OTHER INFORMATION: Description of Artificial Sequence: gp140.modSF162<400> SEQUENCE: 7gaattcgcca ccatggatgc aatgaagaga gggctctgct gtgtgctgct gctgtgtgga 60gcagtcttcg tttcgcccag cgccgtggag aagctgtggg tgaccgtgta ctacggcgtg 120cccgtgtgga aggaggccac caccaccctg ttctgcgcca gcgacgccaa ggcctacgac 180accgaggtgc acaacgtgtg ggccacccac gcctgcgtgc ccaccgaccc caacccccag 240gagatcgtgc tggagaacgt gaccgagaac ttcaacatgt ggaagaacaa catggtggag 300cagatgcacg aggacatcat cagcctgtgg gaccagagcc tgaagccctg cgtgaagctg 360acccccctgt gcgtgaccct gcactgcacc aacctgaaga acgccaccaa caccaagagc 420agcaactgga aggagatgga ccgcggcgag atcaagaact gcagcttcaa ggtgaccacc 480agcatccgca acaagatgca gaaggagtac gccctgttct acaagctgga cgtggtgccc 540atcgacaacg acaacaccag ctacaagctg atcaactgca acaccagcgt gatcacccag 600gcctgcccca aggtgagctt cgagcccatc cccatccact actgcgcccc cgccggcttc 660gccatcctga agtgcaacga caagaagttc aacggcagcg gcccctgcac caacgtgagc 720accgtgcagt gcacccacgg catccgcccc gtggtgagca cccagctgct gctgaacggc 780agcctggccg aggagggcgt ggtgatccgc agcgagaact tcaccgacaa cgccaagacc 840atcatcgtgc agctgaagga gagcgtggag atcaactgca cccgccccaa caacaacacc 900cgcaagagca tcaccatcgg ccccggccgc gccttctacg ccaccggcga catcatcggc 960gacatccgcc aggcccactg caacatcagc ggcgagaagt ggaacaacac cctgaagcag 1020atcgtgacca agctgcaggc ccagttcggc aacaagacca tcgtgttcaa gcagagcagc 1080ggcggcgacc ccgagatcgt gatgcacagc ttcaactgcg gcggcgagtt cttctactgc 1140aacagcaccc agctgttcaa cagcacctgg aacaacacca tcggccccaa caacaccaac 1200ggcaccatca ccctgccctg ccgcatcaag cagatcatca accgctggca ggaggtgggc 1260aaggccatgt acgccccccc catccgcggc cagatccgct gcagcagcaa catcaccggc 1320ctgctgctga cccgcgacgg cggcaaggag atcagcaaca ccaccgagat cttccgcccc 1380ggcggcggcg acatgcgcga caactggcgc agcgagctgt acaagtacaa ggtggtgaag 1440atcgagcccc tgggcgtggc ccccaccaag gccaagcgcc gcgtggtgca gcgcgagaag 1500cgcgccgtga ccctgggcgc catgttcctg ggcttcctgg gcgccgccgg cagcaccatg 1560ggcgcccgca gcctgaccct gaccgtgcag gcccgccagc tgctgagcgg catcgtgcag 1620cagcagaaca acctgctgcg cgccatcgag gcccagcagc acctgctgca gctgaccgtg 1680tggggcatca agcagctgca ggcccgcgtg ctggccgtgg agcgctacct gaaggaccag 1740cagctgctgg gcatctgggg ctgcagcggc aagctgatct gcaccaccgc cgtgccctgg 1800aacgccagct ggagcaacaa gagcctggac cagatctgga acaacatgac ctggatggag 1860tgggagcgcg agatcgacaa ctacaccaac ctgatctaca ccctgatcga ggagagccag 1920aaccagcagg agaagaacga gcaggagctg ctggagctgg acaagtgggc cagcctgtgg 1980aactggttcg acatcagcaa gtggctgtgg tacatctaac tcgag 2025 <210> SEQ ID NO 8<211> LENGTH: 1944 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence<220> FEATURE: <223>OTHER INFORMATION: Description of Artificial Sequence:gp140.modSF162.delV2 <400> SEQUENCE 8gaattcgcca ccatggatgc aatgaagaga gggctctgct gtgtgctgct gctgtgtgga 60gcagtcttcg tttcgcccag cgccgtggag aagctgtggg tgaccgtgta ctacggcgtg 120cccgtgtgga aggaggccac caccaccctg ttctgcgcca gcgacgccaa ggcctacgac 180accgaggtgc acaacgtgtg ggccacccac gcctgcgtgc ccaccgaccc caacccccag 240gagatcgtgc tggagaacgt gaccgagaac ttcaacatgt ggaagaacaa catggtggag 300cagatgcacg aggacatcat cagcctgtgg gaccagagcc tgaagccctg cgtgaagctg 360acccccctgt gcgtgaccct gcactgcacc aacctgaaga acgccaccaa caccaagagc 420agcaactgga aggagatgga ccgcggcgag atcaagaact gcagcttcaa ggtgggcgcc 480ggcaagctga tcaactgcaa caccagcgtg atcacccagg cctgccccaa ggtgagcttc 540gagcccatcc ccatccacta ctgcgccccc gccggcttcg ccatcctgaa gtgcaacgac 600aagaagttca acggcagcgg cccctgcacc aacgtgagca ccgtgcagtg cacccacggc 660atccgccccg tggtgagcac ccagctgctg ctgaacggca gcctggccga ggagggcgtg 720gtgatccgca gcgagaactt caccgacaac gccaagacca tcatcgtgca gctgaaggag 780agcgtggaga tcaactgcac ccgccccaac aacaacaccc gcaagagcat caccatcggc 840cccggccgcg ccttctacgc caccggcgac atcatcggcg acatccgcca ggcccactgc 900aacatcagcg gcgagaagtg gaacaacacc ctgaagcaga tcgtgaccaa gctgcaggcc 960cagttcggca acaagaccat cgtgttcaag cagagcagcg gcggcgaccc cgagatcgtg 1020atgcacagct tcaactgcgg cggcgagttc ttctactgca acagcaccca gctgttcaac 1080agcacctgga acaacaccat cggccccaac aacaccaacg gcaccatcac cctgccctgc 1140cgcatcaagc agatcatcaa ccgctggcag gaggtgggca aggccatgta cgcccccccc 1200atccgcggcc agatccgctg cagcagcaac atcaccggcc tgctgctgac ccgcgacggc 1260ggcaaggaga tcagcaacac caccgagatc ttccgccccg gcggcggcga catgcgcgac 1320aactggcgca gcgagctgta caagtacaag gtggtgaaga tcgagcccct gggcgtggcc 1380cccaccaagg ccaagcgccg cgtggtgcag cgcgagaagc gcgccgtgac cctgggcgcc 1440atgttcctgg gcttcctggg cgccgccggc agcaccatgg gcgcccgcag cctgaccctg 1500accgtgcagg cccgccagct gctgagcggc atcgtgcagc agcagaacaa cctgctgcgc 1560gccatcgagg cccagcagca cctgctgcag ctgaccgtgt ggggcatcaa gcagctgcag 1620gcccgcgtgc tggccgtgga gcgctacctg aaggaccagc agctgctggg catctggggc 1680tgcagcggca agctgatctg caccaccgcc gtgccctgga acgccagctg gagcaacaag 1740agcctggacc agatctggaa caacatgacc tggatggagt gggagcgcga gatcgacaac 1800tacaccaacc tgatctacac cctgatcgag gagagccaga accagcagga gaagaacgag 1860caggagctgc tggagctgga caagtgggcc agcctgtgga actggttcga catcagcaag 1920tggctgtggt acatctaact cgag 1944 <210> SEQ ID NO 9 <211> LENGTH: 1944<212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223>OTHER INFORMATION: Description of Artificial Sequence:gp140.modSF162.delV1/V2 <400> SEQUENCE: 9gaattcgcca ccatggatgc aatgaagaga gggctctgct gtgtgctgct gctgtgtgga 60gcagtcttcg tttcgcccag cgccgtggag aagctgtggg tgaccgtgta ctacggcgtg 120cccgtgtgga aggaggccac caccaccctg ttctgcgcca gcgacgccaa ggcctacgac 180accgaggtgc acaacgtgtg ggccacccac gcctgcgtgc ccaccgaccc caacccccag 240gagatcgtgc tggagaacgt gaccgagaac ttcaacatgt ggaagaacaa catggtggag 300cagatgcacg aggacatcat cagcctgtgg gaccagagcc tgaagccctg cgtgaagctg 360acccccctgt gcgtgaccct gcactgcacc aacctgaaga acgccaccaa caccaagagc 420agcaactgga aggagatgga ccgcggcgag atcaagaact gcagcttcaa ggtgggcgcc 480ggcaagctga tcaactgcaa caccagcgtg atcacccagg cctgccccaa ggtgagcttc 540gagcccatcc ccatccacta ctgcgccccc gccggcttcg ccatcctgaa gtgcaacgac 600aagaagttca acggcagcgg cccctgcacc aacgtgagca ccgtgcagtg cacccacggc 660atccgccccg tggtgagcac ccagctgctg ctgaacggca gcctggccga ggagggcgtg 720gtgatccgca gcgagaactt caccgacaac gccaagacca tcatcgtgca gctgaaggag 780agcgtggaga tcaactgcac ccgccccaac aacaacaccc gcaagagcat caccatcggc 840cccggccgcg ccttctacgc caccggcgac atcatcggcg acatccgcca ggcccactgc 900aacatcagcg gcgagaagtg gaacaacacc ctgaagcaga tcgtgaccaa gctgcaggcc 960cagttcggca acaagaccat cgtgttcaag cagagcagcg gcggcgaccc cgagatcgtg 1020atgcacagct tcaactgcgg cggcgagttc ttctactgca acagcaccca gctgttcaac 1080agcacctgga acaacaccat cggccccaac aacaccaacg gcaccatcac cctgccctgc 1140cgcatcaagc agatcatcaa ccgctggcag gaggtgggca aggccatgta cgcccccccc 1200atccgcggcc agatccgctg cagcagcaac atcaccggcc tgctgctgac ccgcgacggc 1260ggcaaggaga tcagcaacac caccgagatc ttccgccccg gcggcggcga catgcgcgac 1320aactggcgca gcgagctgta caagtacaag gtggtgaaga tcgagcccct gggcgtggcc 1380cccaccaagg ccaagcgccg cgtggtgcag cgcgagaagc gcgccgtgac cctgggcgcc 1440atgttcctgg gcttcctggg cgccgccggc agcaccatgg gcgcccgcag cctgaccctg 1500accgtgcagg cccgccagct gctgagcggc atcgtgcagc agcagaacaa cctgctgcgc 1560gccatcgagg cccagcagca cctgctgcag ctgaccgtgt ggggcatcaa gcagctgcag 1620gcccgcgtgc tggccgtgga gcgctacctg aaggaccagc agctgctggg catctggggc 1680tgcagcggca agctgatctg caccaccgcc gtgccctgga acgccagctg gagcaacaag 1740agcctggacc agatctggaa caacatgacc tggatggagt gggagcgcga gatcgacaac 1800tacaccaacc tgatctacac cctgatcgag gagagccaga accagcagga gaagaacgag 1860caggagctgc tggagctgga caagtgggcc agcctgtgga actggttcga catcagcaag 1920tggctgtggt acatctaact cgag 1944 <210> SEQ ID NO 10 <211> LENGTH: 2025<212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223>OTHER INFORMATION: Description of Artificial Sequence:gp140.mut.modSF162 <400> SEQUENCE: 10gaattcgcca ccatggatgc aatgaagaga gggctctgct gtgtgctgct gctgtgtgga 60gcagtcttcg tttcgcccag cgccgtggag aagctgtggg tgaccgtgta ctacggcgtg 120cccgtgtgga aggaggccac caccaccctg ttctgcgcca gcgacgccaa ggcctacgac 180accgaggtgc acaacgtgtg ggccacccac gcctgcgtgc ccaccgaccc caacccccag 240gagatcgtgc tggagaacgt gaccgagaac ttcaacatgt ggaagaacaa catggtggag 300cagatgcacg aggacatcat cagcctgtgg gaccagagcc tgaagccctg cgtgaagctg 360acccccctgt gcgtgaccct gcactgcacc aacctgaaga acgccaccaa caccaagagc 420agcaactgga aggagatgga ccgcggcgag atcaagaact gcagcttcaa ggtgaccacc 480agcatccgca acaagatgca gaaggagtac gccctgttct acaagctgga cgtggtgccc 540atcgacaacg acaacaccag ctacaagctg atcaactgca acaccagcgt gatcacccag 600gcctgcccca aggtgagctt cgagcccatc cccatccact actgcgcccc cgccggcttc 660gccatcctga agtgcaacga caagaagttc aacggcagcg gcccctgcac caacgtgagc 720accgtgcagt gcacccacgg catccgcccc gtggtgagca cccagctgct gctgaacggc 780agcctggccg aggagggcgt ggtgatccgc agcgagaact tcaccgacaa cgccaagacc 840atcatcgtgc agctgaagga gagcgtggag atcaactgca cccgccccaa caacaacacc 900cgcaagagca tcaccatcgg ccccggccgc gccttctacg ccaccggcga catcatcggc 960gacatccgcc aggcccactg caacatcagc ggcgagaagt ggaacaacac cctgaagcag 1020atcgtgacca agctgcaggc ccagttcggc aacaagacca tcgtgttcaa gcagagcagc 1080ggcggcgacc ccgagatcgt gatgcacagc ttcaactgcg gcggcgagtt cttctactgc 1140aacagcaccc agctgttcaa cagcacctgg aacaacacca tcggccccaa caacaccaac 1200ggcaccatca ccctgccctg ccgcatcaag cagatcatca accgctggca ggaggtgggc 1260aaggccatgt acgccccccc catccgcggc cagatccgct gcagcagcaa catcaccggc 1320ctgctgctga cccgcgacgg cggcaaggag atcagcaaca ccaccgagat cttccgcccc 1380ggcggcggcg acatgcgcga caactggcgc agcgagctgt acaagtacaa ggtggtgaag 1440atcgagcccc tgggcgtggc ccccaccaag gccaagcgcc gcgtggtgca gcgcgagaag 1500agcgccgtga ccctgggcgc catgttcctg ggcttcctgg gcgccgccgg cagcaccatg 1560ggcgcccgca gcctgaccct gaccgtgcag gcccgccagc tgctgagcgg catcgtgcag 1620cagcagaaca acctgctgcg cgccatcgag gcccagcagc acctgctgca gctgaccgtg 1680tggggcatca agcagctgca ggcccgcgtg ctggccgtgg agcgctacct gaaggaccag 1740cagctgctgg gcatctgggg ctgcagcggc aagctgatct gcaccaccgc cgtgccctgg 1800aacgccagct ggagcaacaa gagcctggac cagatctgga acaacatgac ctggatggag 1860tgggagcgcg agatcgacaa ctacaccaac ctgatctaca ccctgatcga ggagagccag 1920aaccagcagg agaagaacga gcaggagctg ctggagctgg acaagtgggc cagcctgtgg 1980aactggttcg acatcagcaa gtggctgtgg tacatctaac tcgag 2025 <210>SEQ ID NO 11 <211> LENGTH: 1944 <212> TYPE: DNA <213>ORGANISM: Artificial Sequence <220> FEATURE: <223>OTHER INFORMATION: Description of Artificial Sequence:gp140.mut.modSF162.delV2 <400> SEQUENCE: 11gaattcgcca ccatggatgc aatgaagaga gggctctgct gtgtgctgct gctgtgtgga 60gcagtcttcg tttcgcccag cgccgtggag aagctgtggg tgaccgtgta ctacggcgtg 120cccgtgtgga aggaggccac caccaccctg ttctgcgcca gcgacgccaa ggcctacgac 180accgaggtgc acaacgtgtg ggccacccac gcctgcgtgc ccaccgaccc caacccccag 240gagatcgtgc tggagaacgt gaccgagaac ttcaacatgt ggaagaacaa catggtggag 300cagatgcacg aggacatcat cagcctgtgg gaccagagcc tgaagccctg cgtgaagctg 360acccccctgt gcgtgaccct gcactgcacc aacctgaaga acgccaccaa caccaagagc 420agcaactgga aggagatgga ccgcggcgag atcaagaact gcagcttcaa ggtgggcgcc 480ggcaagctga tcaactgcaa caccagcgtg atcacccagg cctgccccaa ggtgagcttc 540gagcccatcc ccatccacta ctgcgccccc gccggcttcg ccatcctgaa gtgcaacgac 600aagaagttca acggcagcgg cccctgcacc aacgtgagca ccgtgcagtg cacccacggc 660atccgccccg tggtgagcac ccagctgctg ctgaacggca gcctggccga ggagggcgtg 720gtgatccgca gcgagaactt caccgacaac gccaagacca tcatcgtgca gctgaaggag 780agcgtggaga tcaactgcac ccgccccaac aacaacaccc gcaagagcat caccatcggc 840cccggccgcg ccttctacgc caccggcgac atcatcggcg acatccgcca ggcccactgc 900aacatcagcg gcgagaagtg gaacaacacc ctgaagcaga tcgtgaccaa gctgcaggcc 960cagttcggca acaagaccat cgtgttcaag cagagcagcg gcggcgaccc cgagatcgtg 1020atgcacagct tcaactgcgg cggcgagttc ttctactgca acagcaccca gctgttcaac 1080agcacctgga acaacaccat cggccccaac aacaccaacg gcaccatcac cctgccctgc 1140cgcatcaagc agatcatcaa ccgctggcag gaggtgggca aggccatgta cgcccccccc 1200atccgcggcc agatccgctg cagcagcaac atcaccggcc tgctgctgac ccgcgacggc 1260ggcaaggaga tcagcaacac caccgagatc ttccgccccg gcggcggcga catgcgcgac 1320aactggcgca gcgagctgta caagtacaag gtggtgaaga tcgagcccct gggcgtggcc 1380cccaccaagg ccaagcgccg cgtggtgcag cgcgagaaga gcgccgtgac cctgggcgcc 1440atgttcctgg gcttcctggg cgccgccggc agcaccatgg gcgcccgcag cctgaccctg 1500accgtgcagg cccgccagct gctgagcggc atcgtgcagc agcagaacaa cctgctgcgc 1560gccatcgagg cccagcagca cctgctgcag ctgaccgtgt ggggcatcaa gcagctgcag 1620gcccgcgtgc tggccgtgga gcgctacctg aaggaccagc agctgctggg catctggggc 1680tgcagcggca agctgatctg caccaccgcc gtgccctgga acgccagctg gagcaacaag 1740agcctggacc agatctggaa caacatgacc tggatggagt gggagcgcga gatcgacaac 1800tacaccaacc tgatctacac cctgatcgag gagagccaga accagcagga gaagaacgag 1860caggagctgc tggagctgga caagtgggcc agcctgtgga actggttcga catcagcaag 1920tggctgtggt acatctaact cgag 1944 <210> SEQ ID NO 12 <211> LENGTH: 1836<212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223>OTHER INFORMATION: Description of Artificial Sequence:gp140.mut.modSF162.delV1/V2 <400> SEQUENCE: 12gaattcgcca ccatggatgc aatgaagaga gggctctgct gtgtgctgct gctgtgtgga 60gcagtcttcg tttcgcccag cgccgtggag aagctgtggg tgaccgtgta ctacggcgtg 120cccgtgtgga aggaggccac caccaccctg ttctgcgcca gcgacgccaa ggcctacgac 180accgaggtgc acaacgtgtg ggccacccac gcctgcgtgc ccaccgaccc caacccccag 240gagatcgtgc tggagaacgt gaccgagaac ttcaacatgt ggaagaacaa catggtggag 300cagatgcacg aggacatcat cagcctgtgg gaccagagcc tgaagccctg cgtgaagctg 360acccccctgt gcgtgggcgc cggcaactgc cagaccagcg tgatcaccca ggcctgcccc 420aaggtgagct tcgagcccat ccccatccac tactgcgccc ccgccggctt cgccatcctg 480aagtgcaacg acaagaagtt caacggcagc ggcccctgca ccaacgtgag caccgtgcag 540tgcacccacg gcatccgccc cgtggtgagc acccagctgc tgctgaacgg cagcctggcc 600gaggagggcg tggtgatccg cagcgagaac ttcaccgaca acgccaagac catcatcgtg 660cagctgaagg agagcgtgga gatcaactgc acccgcccca acaacaacac ccgcaagagc 720atcaccatcg gccccggccg cgccttctac gccaccggcg acatcatcgg cgacatccgc 780caggcccact gcaacatcag cggcgagaag tggaacaaca ccctgaagca gatcgtgacc 840aagctgcagg cccagttcgg caacaagacc atcgtgttca agcagagcag cggcggcgac 900cccgagatcg tgatgcacag cttcaactgc ggcggcgagt tcttctactg caacagcacc 960cagctgttca acagcacctg gaacaacacc atcggcccca acaacaccaa cggcaccatc 1020accctgccct gccgcatcaa gcagatcatc aaccgctggc aggaggtggg caaggccatg 1080tacgcccccc ccatccgcgg ccagatccgc tgcagcagca acatcaccgg cctgctgctg 1140acccgcgacg gcggcaagga gatcagcaac accaccgaga tcttccgccc cggcggcggc 1200gacatgcgcg acaactggcg cagcgagctg tacaagtaca aggtggtgaa gatcgagccc 1260ctgggcgtgg cccccaccaa ggccaagcgc cgcgtggtgc agcgcgagaa gagcgccgtg 1320accctgggcg ccatgttcct gggcttcctg ggcgccgccg gcagcaccat gggcgcccgc 1380agcctgaccc tgaccgtgca ggcccgccag ctgctgagcg gcatcgtgca gcagcagaac 1440aacctgctgc gcgccatcga ggcccagcag cacctgctgc agctgaccgt gtggggcatc 1500aagcagctgc aggcccgcgt gctggccgtg gagcgctacc tgaaggacca gcagctgctg 1560ggcatctggg gctgcagcgg caagctgatc tgcaccaccg ccgtgccctg gaacgccagc 1620tggagcaaca agagcctgga ccagatctgg aacaacatga cctggatgga gtgggagcgc 1680gagatcgaca actacaccaa cctgatctac accctgatcg aggagagcca gaaccagcag 1740gagaagaacg agcaggagct gctggagctg gacaagtggg ccagcctgtg gaactggttc 1800gacatcagca agtggctgtg gtacatctaa ctcgag 1836 <210> SEQ ID NO 13 <211>LENGTH: 2025 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220>FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence:gp140.mut7.modSF162 <400> SEQUENCE: 13gaattcgcca ccatggatgc aatgaagaga gggctctgct gtgtgctgct gctgtgtgga 60gcagtcttcg tttcgcccag cgccgtggag aagctgtggg tgaccgtgta ctacggcgtg 120cccgtgtgga aggaggccac caccaccctg ttctgcgcca gcgacgccaa ggcctacgac 180accgaggtgc acaacgtgtg ggccacccac gcctgcgtgc ccaccgaccc caacccccag 240gagatcgtgc tggagaacgt gaccgagaac ttcaacatgt ggaagaacaa catggtggag 300cagatgcacg aggacatcat cagcctgtgg gaccagagcc tgaagccctg cgtgaagctg 360acccccctgt gcgtgaccct gactgcacc aacctgaaga acgccaccaa caccaagagc 420agcaactgga aggagatgga ccgcggcgag atcaagaact gcagcttcaa ggtgaccacc 480agcatccgca acaagatgca gaaggagtac gccctgttct acaagctgga cgtggtgccc 540atcgacaacg acaacaccag ctacaagctg atcaactgca acaccagcgt gatcacccag 600gcctgcccca aggtgagctt cgagcccatc cccatccact actgcgcccc cgccggcttc 660gccatcctga agtgcaacga caagaagttc aacggcagcg gcccctgcac caacgtgagc 720accgtgcagt gcacccacgg catccgcccc gtggtgagca cccagctgct gctgaacggc 780agcctggccg aggagggcgt ggtgatccgc agcgagaact tcaccgacaa cgccaagacc 840atcatcgtgc agctgaagga gagcgtggag atcaactgca cccgccccaa caacaacacc 900cgcaagagca tcaccatcgg ccccggccgc gccttctacg ccaccggcga catcatcggc 960gacatccgcc aggcccactg caacatcagc ggcgagaagt ggaacaacac cctgaagcag 1020atcgtgacca agctgcaggc ccagttcggc aacaagacca tcgtgttcaa gcagagcagc 1080ggcggcgacc ccgagatcgt gatgcacagc ttcaactgcg gcggcgagtt cttctactgc 1140aacagcaccc agctgttcaa cagcacctgg aacaacacca tcggccccaa caacaccaac 1200ggcaccatca ccctgccctg ccgcatcaag cagatcatca accgctggca ggaggtgggc 1260aaggccatgt acgccccccc catccgcggc cagatccgct gcagcagcaa catcaccggc 1320ctgctgctga cccgcgacgg cggcaaggag atcagcaaca ccaccgagat cttccgcccc 1380ggcggcggcg acatgcgcga caactggcgc agcgagctgt acaagtacaa ggtggtgaag 1440atcgagcccc tgggcgtggc ccccaccaag gccatcagca gcgtggtgca gagcgagaag 1500agcgccgtga ccctgggcgc catgttcctg ggcttcctgg gcgccgccgg cagcaccatg 1560ggcgcccgca gcctgaccct gaccgtgcag gcccgccagc tgctgagcgg catcgtgcag 1620cagcagaaca acctgctgcg cgccatcgag gcccagcagc acctgctgca gctgaccgtg 1680tggggcatca agcagctgca ggcccgcgtg ctggccgtgg agcgctacct gaaggaccag 1740cagctgctgg gcatctgggg ctgcagcggc aagctgatct gcaccaccgc cgtgccctgg 1800aacgccagct ggagcaacaa gagcctggac cagatctgga acaacatgac ctggatggag 1860tgggagcgcg agatcgacaa ctacaccaac ctgatctaca ccctgatcga ggagagccag 1920aaccagcagg agaagaacga gcaggagctg ctggagctgg acaagtgggc cagcctgtgg 1980aactggttcg acatcagcaa gtggctgtgg tacatctaac tcgag 2025 <210>SEQ ID NO 14 <211> LENGHT: 1944 <212> TYPE: DNA <213>ORGANISM: Artificial Sequence <220> FEATURE: <223>OTHER INFORMATION: Description of Artificial Sequence:gp140.mut7.modSF162.delV2 <400> SEQUENCE: 14gaattcgcca ccatggatgc aatgaagaga gggctctgct gtgtgctgct gctgtgtgga 60gcagtcttcg tttcgcccag cgccgtggag aagctgtggg tgaccgtgta ctacggcgtg 120cccgtgtgga aggaggccac caccaccctg ttctgcgcca gcgacgccaa ggcctacgac 180accgaggtgc acaacgtgtg ggccacccac gcctgcgtgc ccaccgaccc caacccccag 240gagatcgtgc tggagaacgt gaccgagaac ttcaacatgt ggaagaacaa catggtggag 300cagatgcacg aggacatcat cagcctgtgg gaccagagcc tgaagccctg cgtgaagctg 360acccccctgt gcgtgaccct gcactgcacc aacctgaaga acgccaccaa caccaagagc 420agcaactgga aggagatgga ccgcggcgag atcaagaact gcagcttcaa ggtgggcgcc 480ggcaagctga tcaactgcaa caccagcgtg atcacccagg cctgccccaa ggtgagcttc 540gagcccatcc ccatccacta ctgcgccccc gccggcttcg ccatcctgaa gtgcaacgac 600aagaagttca acggcagcgg cccctgcacc aacgtgagca ccgtgcagtg cacccacggc 660atccgccccg tggtgagcac ccagctgctg ctgaacggca gcctggccga ggagggcgtg 720gtgatccgca gcgagaactt caccgacaac gccaagacca tcatcgtgca gctgaaggag 780agcgtggaga tcaactgcac ccgccccaac aacaacaccc gcaagagcat caccatcggc 840cccggccgcg ccttctacgc caccggcgac atcatcggcg acatccgcca ggcccactgc 900aacatcagcg gcgagaagtg gaacaacacc ctgaagcaga tcgtgaccaa gctgcaggcc 960cagttcggca acaagaccat cgtgttcaag cagagcagcg gcggcgaccc cgagatcgtg 1020atgcacagct tcaactgcgg cggcgagttc ttctactgca acagcaccca gctgttcaac 1080agcacctgga acaacaccat cggccccaac aacaccaacg gcaccatcac cctgccctgc 1140cgcatcaagc agatcatcaa ccgctggcag gaggtgggca aggccatgta cgcccccccc 1200atccgcggcc agatccgctg cagcagcaac atcaccggcc tgctgctgac ccgcgacggc 1260ggcaaggaga tcagcaacac caccgagatc ttccgccccg gcggcggcga catgcgcgac 1320aactggcgca gcgagctgta caagtacaag gtggtgaaga tcgagcccct gggcgtggcc 1380cccaccaagg ccatcagcag cgtggtgcag agcgagaaga gcgccgtgac cctgggcgcc 1440atgttcctgg gcttcctggg cgccgccggc agcaccatgg gcgcccgcag cctgaccctg 1500accgtgcagg cccgccagct gctgagcggc atcgtgcagc agcagaacaa cctgctgcgc 1560gccatcgagg cccagcagca cctgctgcag ctgaccgtgt ggggcatcaa gcagctgcag 1620gcccgcgtgc tggccgtgga gcgctacctg aaggaccagc agctgctggg catctggggc 1680tgcagcggca agctgatctg caccaccgcc gtgccctgga acgccagctg gagcaacaag 1740agcctggacc agatctggaa caacatgacc tggatggagt gggagcgcga gatcgacaac 1800tacaccaacc tgatctacac cctgatcgag gagagccaga accagcagga gaagaacgag 1860caggagctgc tggagctgga caagtgggcc agcctgtgga actggttcga catcagcaag 1920tggctgtggt acatctaact cgag 1944 <210> SEQ ID NO 15 <211> LENGTH: 1836<212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223>OTHER INFORMATION: Description of Artificial Sequence:gp140.mut7.modSF162.delV1/V2 <400> SEQUENCE: 15gaattcgcca ccatggatgc aatgaagaga gggctctgct gtgtgctgct gctgtgtgga 60gcagtcttcg tttcgcccag cgccgtggag aagctgtggg tgaccgtgta ctacggcgtg 120cccgtgtgga aggaggccac caccaccctg ttctgcgcca gcgacgccaa ggcctacgac 180accgaggtgc acaacgtgtg ggccacccac gcctgcgtgc ccaccgaccc caacccccag 240gagatcgtgc tggagaacgt gaccgagaac ttcaacatgt ggaagaacaa catggtggag 300cagatgcacg aggacatcat cagcctgtgg gaccagagcc tgaagccctg cgtgaagctg 360acccccctgt gcgtgggcgc cggcaactgc cagaccagcg tgatcaccca ggcctgcccc 420aaggtgagct tcgagcccat ccccatccac tactgcgccc ccgccggctt cgccatcctg 480aagtgcaacg acaagaagtt caacggcagc ggcccctgca ccaacgtgag caccgtgcag 540tgcacccacg gcatccgccc cgtggtgagc acccagctgc tgctgaacgg cagcctggcc 600gaggagggcg tggtgatccg cagcgagaac ttcaccgaca acgccaagac catcatcgtg 660cagctgaagg agagcgtgga gatcaactgc acccgcccca acaacaacac ccgcaagagc 720atcaccatcg gccccggccg cgccttctac gccaccggcg acatcatcgg cgacatccgc 780caggcccact gcaacatcag cggcgagaag tggaacaaca ccctgaagca gatcgtgacc 840aagctgcagg cccagttcgg caacaagacc atcgtgttca agcagagcag cggcggcgac 900cccgagatcg tgatgcacag cttcaactgc ggcggcgagt tcttctactg caacagcacc 960cagctgttca acagcacctg gaacaacacc atcggcccca acaacaccaa cggcaccatc 1020accctgccct gccgcatcaa gcagatcatc aaccgctggc aggaggtggg caaggccatg 1080tacgcccccc ccatccgcgg ccagatccgc tgcagcagca acatcaccgg cctgctgctg 1140acccgcgacg gcggcaagga gatcagcaac accaccgaga tcttccgccc cggcggcggc 1200gacatgcgcg acaactggcg cagcgagctg tacaagtaca aggtggtgaa gatcgagccc 1260ctgggcgtgg cccccaccaa ggccatcagc agcgtggtgc agagcgagaa gagcgccgtg 1320accctgggcg ccatgttcct gggcttcctg ggcgccgccg gcagcaccat gggcgcccgc 1380agcctgaccc tgaccgtgca ggcccgccag ctgctgagcg gcatcgtgca gcagcagaac 1440aacctgctgc gcgccatcga ggcccagcag cacctgctgc agctgaccgt gtggggcatc 1500aagcagctgc aggcccgcgt gctggccgtg gagcgctacc tgaaggacca gcagctgctg 1560ggcatctggg gctgcagcgg caagctgatc tgcaccaccg ccgtgccctg gaacgccagc 1620tggagcaaca agagcctgga ccagatctgg aacaacatga cctggatgga gtgggagcgc 1680gagatcgaca actacaccaa cctgatctac accctgatcg aggagagcca gaaccagcag 1740gagaagaacg agcaggagct gctggagctg gacaagtggg ccagcctgtg gaactggttc 1800gacatcagca agtggctgtg gtacatctaa ctcgag 1836 <210> SEQ ID NO 16 <211>LENGTH: 2025 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220>FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence:gp140.mut8.modSF162 <400> SEQUENCE: 16gaattcgcca ccatggatgc aatgaagaga gggctctgct gtgtgctgct gctgtgtgga 60gcagtcttcg tttcgcccag cgccgtggag aagctgtggg tgaccgtgta ctacggcgtg 120cccgtgtgga aggaggccac caccaccctg ttctgcgcca gcgacgccaa ggcctacgac 180accgaggtgc acaacgtgtg ggccacccac gcctgcgtgc ccaccgaccc caacccccag 240gagatcgtgc tggagaacgt gaccgagaac ttcaacatgt ggaagaacaa catggtggag 300cagatgcacg aggacatcat cagcctgtgg gaccagagcc tgaagccctg cgtgaagctg 360acccccctgt gcgtgaccct gcactgcacc aacctgaaga acgccaccaa caccaagagc 420agcaactgga aggagatgga ccgcggcgag atcaagaact gcagcttcaa ggtgaccacc 480agcatccgca acaagatgca gaaggagtac gccctgttct acaagctgga cgtggtgccc 540atcgacaacg acaacaccag ctacaagctg atcaactgca acaccagcgt gatcacccag 600gcctgcccca aggtgagctt cgagcccatc cccatccact actgcgcccc cgccggcttc 660gccatcctga agtgcaacga caagaagttc aacggcagcg gcccctgcac caacgtgagc 720accgtgcagt gcacccacgg catccgcccc gtggtgagca cccagctgct gctgaacggc 780agcctggccg aggagggcgt ggtgatccgc agcgagaact tcaccgacaa cgccaagacc 840atcatcgtgc agctgaagga gagcgtggag atcaactgca cccgccccaa caacaacacc 900cgcaagagca tcaccatcgg ccccggccgc gccttctacg ccaccggcga catcatcggc 960gacatccgcc aggcccactg caacatcagc ggcgagaagt ggaacaacac cctgaagcag 1020atcgtgacca agctgcaggc ccagttcggc aacaagacca tcgtgttcaa gcagagcagc 1080ggcggcgacc ccgagatcgt gatgcacagc ttcaactgcg gcggcgagtt cttctactgc 1140aacagcaccc agctgttcaa cagcacctgg aacaacacca tcggccccaa caacaccaac 1200ggcaccatca ccctgccctg ccgcatcaag cagatcatca accgctggca ggaggtgggc 1260aaggccatgt acgccccccc catccgcggc cagatccgct gcagcagcaa catcaccggc 1320ctgctgctga cccgcgacgg cggcaaggag atcagcaaca ccaccgagat cttccgcccc 1380ggcggcggcg acatgcgcga caactggcgc agcgagctgt acaagtacaa ggtggtgaag 1440atcgagcccc tgggcgtggc ccccaccatc gccatcagca gcgtggtgca gagcgagaag 1500agcgccgtga ccctgggcgc catgttcctg ggcttcctgg gcgccgccgg cagcacctag 1560ggcgcccgca gcctgaccct gaccgtgcag gcccgccagc tgctgagcgg catcgtgcag 1620cagcagaaca acctgctgcg cgccatcgag gcccagcagc acctgctgca gctgaccgtg 1680tggggcatca agcagctgca ggcccgcgtg ctggccgtgg agcgctacct gaaggaccag 1740cagctgctgg gcatctgggg ctgcagcggc aagctgatct gcaccaccgc cgtgccctgg 1800aacgccagct ggagcaacaa gagcctggac cagatctgga acaacatgac ctggatggag 1860tgggagcgcg agatcgacaa ctacaccaac ctgatctaca ccctgatcga ggagagccag 1920aaccagcagg agaagaacga gcaggagctg ctggagctgg acaagtgggc cagcctgtgg 1980aactggttcg acatcagcaa gtggctgtgg tacatctaac tcgag 2025 <210>SEQ ID NO 17 <211> LENGTH: 1944 <212> TYPE: DNA <213>ORGANISM: Artificial Sequence <220> FEATURE: <223>OTHER INFORMATION: Description of Artificial Sequence:gp140.mut8.modSF162.delV2 <400> SEQUENCE: 17gaattcgcca ccatggatgc aatgaagaga gggctctgct gtgtgctgct gctgtgtgga 60gcagtcttcg tttcgcccag cgccgtggag aagctgtggg tgaccgtgta ctacggcgtg 120cccgtgtgga aggaggccac caccaccctg ttctgcgcca gcgacgccaa ggcctacgac 180accgaggtgc acaacgtgtg ggccacccac gcctgcgtgc ccaccgaccc caacccccag 240gagatcgtgc tggagaacgt gaccgagaac ttcaacatgt ggaagaacaa catggtggag 300cagatgcacg aggacatcat cagcctgtgg gaccagagcc tgaagccctg cgtgaagctg 360acccccctgt gcgtgaccct gcactgcacc aacctgaaga acgccaccaa caccaagagc 420agcaactgga aggagatgga ccgcggcgag atcaagaact gcagcttcaa ggtgggcgcc 480ggcaagctga tcaactgcaa caccagcgtg atcacccagg cctgccccaa ggtgagcttc 540gagcccatcc ccatccacta ctgcgccccc gccggcttcg ccatcctgaa gtgcaacgac 600aagaagttca acggcagcgg cccctgcacc aacgtgagca ccgtgcagtg cacccacggc 660atccgccccg tggtgagcac ccagctgctg ctgaacggca gcctggccga ggagggcgtg 720gtgatccgca gcgagaactt caccgacaac gccaagacca tcatcgtgca gctgaaggag 780agcgtggaga tcaactgcac ccgccccaac aacaacaccc gcaagagcat caccatcggc 840cccggccgcg ccttctacgc caccggcgac atcatcggcg acatccgcca ggcccactgc 900aacatcagcg gcgagaagtg gaacaacacc ctgaagcaga tcgtgaccaa gctgcaggcc 960cagttcggca acaagaccat cgtgttcaag cagagcagcg gcggcgaccc cgagatcgtg 1020atgcacagct tcaactgcgg cggcgagttc ttctactgca acagcaccca gctgttcaac 1080agcacctgga acaacaccat cggccccaac aacaccaacg gcaccatcac cctgccctgc 1140cgcatcaagc agatcatcaa ccgctggcag gaggtgggca aggccatgta cgcccccccc 1200atccgcggcc agatccgctg cagcagcaac atcaccggcc tgctgctgac ccgcgacggc 1260ggcaaggaga tcagcaacac caccgagatc ttccgccccg gcggcggcga catgcgcgac 1320aactggcgca gcgagctgta caagtacaag gtggtgaaga tcgagcccct gggcgtggcc 1380cccaccatcg ccatcagcag cgtggtgcag agcgagaaga gcgccgtgac cctgggcgcc 1440atgttcctgg gcttcctggg cgccgccggc agcaccatgg gcgcccgcag cctgaccctg 1500accgtgcagg cccgccagct gctgagcggc atcgtgcagc agcagaacaa cctgctgcgc 1560gccatcgagg cccagcagca cctgctgcag ctgaccgtgt ggggcatcaa gcagctgcag 1620gcccgcgtgc tggccgtgga gcgctqcctg aaggaccagc agctgctggg catctggggc 1680tgcagcggca agctgatctg caccaccgcc gtgccctgga acgccagctg gagcaacaag 1740agcctggacc agatctggaa caacatgacc tggatggagt gggagcgcga gatcgacaac 1800tacaccaacc tgatctacac cctgatcgag gagagccaga accagcagga gaagaacgag 1860caggagctgc tggagctgga caagtgggcc agcctgtgga actggttcga catcagcaag 1920tggctgtggt acatctaact cgag 1944 <210> SEQ ID NO 18 <211> LENGTH: 1836<212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223>OTHER INFORMATION: Description of Artificial Sequence:gp140.mut8.modSF162.delV1/V2 <400> SEQUENCE: 18gaattcgcca ccatggatgc aatgaagaga gggctctgct gtgtgctgct gctgtgtgga 60gcagtcttcg tttcgcccag cgccgtggag aagctgtggg tgaccgtgta ctacggcgtg 120cccgtgtgga aggaggccac caccaccctg ttctgcgcca gcgacgccaa ggcctacgac 180accgaggtgc acaacgtgtg ggccacccac gcctgcgtgc ccaccgaccc caacccccag 240gagatcgtgc tggagaacgt gaccgagaac ttcaacatgt ggaagaacaa catggtggag 300cagatgcacg aggacatcat cagcctgtgg gaccagagcc tgaagccctg cgtgaagctg 360acccccctgt gcgtgggcgc cggcaactgc cagaccagcg tgatcaccca ggcctgcccc 420aaggtgagct tcgagcccat ccccatccac tactgcgccc ccgccggctt cgccatcctg 480aagtgcaacg acaagaagtt caacggcagc ggcccctgca ccaacgtgag caccgtgcag 540tgcacccacg gcatccgccc cgtggtgagc acccagctgc tgctgaacgg cagcctggcc 600gaggagggcg tggtgatccg cagcgagaac ttcaccgaca acgccaagac catcatcgtg 660cagctgaagg agagcgtgga gatcaactgc acccgcccca acaacaacac ccgcaagagc 720atcaccatcg gccccggccg cgccttctac gccaccggcg acatcatcgg cgacatccgc 780caggcccact gcaacatcag cggcgagaag tggaacaaca ccctgaagca gatcgtgacc 840aagctgcagg cccagttcgg caacaagacc atcgtgttca agcagagcag cggcggcgac 900cccgagatcg tgatgcacag cttcaactgc ggcggcgagt tcttctactg caacagcacc 960cagctgttca acagcacctg gaacaacacc atcggcccca acaacaccaa cggcaccatc 1020accctgccct gccgcatcaa gcagatcatc aaccgctggc aggaggtggg caaggccatg 1080tacgcccccc ccatccgcgg ccagatccgc tgcagcagca acatcaccgg cctgctgctg 1140acccgcgacg gcggcaagga gatcagcaac accaccgaga tcttccgccc cggcggcggc 1200gacatgcgcg acaactggcg cagcgagctg tacaagtaca aggtggtgaa gatcgagccc 1260ctgggcgtgg cccccaccat cgccatcagc agcgtggtgc agagcgagaa gagcgccgtg 1320accctgggcg ccatgttcct gggcttcctg ggcgccgccg gcagcaccat gggcgcccgc 1380agcctgaccc tgaccgtgca ggcccgccag ctgctgagcg gcatcgtgca gcagcagaac 1440aacctgctgc gcgccatcga ggcccagcag cacctgctgc agctgaccgt gtggggcatc 1500aagcagctgc aggcccgcgt gctggccgtg gagcgctacc tgaaggacca gcagctgctg 1560ggcatctggg gctgcagcgg caagctgatc tgcaccaccg ccgtgccctg gaacgccagc 1620tggagcaaca agagcctgga ccagatctgg aacaacatga cctggatgga gtgggagcgc 1680gagatcgaca actacaccaa cctgatctac accctgatcg aggagagcca gaaccagcag 1740gagaagaacg agcaggagct gctggagctg gacaagtggg ccagcctgtg gaactggttc 1800gacatcagca agtggctgtg gtacatctaa ctcgag 1836 <210> SEQ ID NO 19 <211>LENGTH: 2547 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220>FEATURE: <223>OTHER INFORMATION: Description of Artificial Sequence: gp160.modSF162<400> SEQUENCE: 19gaattcgcca ccatggatgc aatgaagaga gggctctgct gtgtgctgct gctgtgtgga 60gcagtcttcg tttcgcccag cgccgtggag aagctgtggg tgaccgtgta ctacggcgtg 120cccgtgtgga aggaggccac caccaccctg ttctgcgcca gcgacgccaa ggcctacgac 180accgaggtgc acaacgtgtg ggccacccac gcctgcgtgc ccaccgaccc caacccccag 240gagatcgtgc tggagaacgt gaccgagaac ttcaacatgt ggaagaacaa catggtggag 300cagatgcacg aggacatcat cagcctgtgg gaccagagcc tgaagccctg cgtgaagctg 360acccccctgt gcgtgaccct gcactgcacc aacctgaaga acgccaccaa caccaagagc 420agcaactgga aggagatgga ccgcggcgag atcaagaact gcagcttcaa ggtgaccacc 480agcatccgca acaagatgca gaaggagtac gccctgttct acaagctgga cgtggtgccc 540atcgacaacg acaacaccag ctacaagctg atcaactgca acaccagcgt gatcacccag 600gcctgcccca aggtgagctt cgagcccatc cccatccact actgcgcccc cgccggcttc 660gccatcctga agtgcaacga caagaagttc aacggcagcg gcccctgcac caacgtgagc 720accgtgcagt gcacccacgg catccgcccc gtggtgagca cccagctgct gctgaacggc 780agcctggccg aggagggcgt ggtgatccgc agcgagaact tcaccgacaa cgccaagacc 840atcatcgtgc agctgaagga gagcgtggag atcaactgca cccgccccaa caacaacacc 900cgcaagagca tcaccatcgg ccccggccgc gccttctacg ccaccggcga catcatcggc 960gacatccgcc aggcccactg caacatcagc ggcgagaagt ggaacaacac cctgaagcag 1020atcgtgacca agctgcaggc ccagttcggc aacaagacca tcgtgttcaa gcagagcagc 1080ggcggcgacc ccgagatcgt gatgcacagc ttcaactgcg gcggcgagtt cttctactgc 1140aacagcaccc agctgttcaa cagcacctgg aacaacacca tcggccccaa caacaccaac 1200ggcaccatca ccctgccctg ccgcatcaag cagatcatca accgctggca ggaggtgggc 1260aaggccatgt acgccccccc catccgcggc cagatccgct gcagcagcaa catcaccggc 1320ctgctgctga cccgcgacgg cggcaaggag atcagcaaca ccaccgagat cttccgcccc 1380ggcggcggcg acatgcgcga caactggcgc agcgagctgt acaagtacaa ggtggtgaag 1440atcgagcccc tgggcgtggc ccccaccaag gccaagcgcc gcgtggtgca gcgcgagaag 1500cgcgccgtga ccctgggcgc catgttcctg ggcttcctgg gcgccgccgg cagcaccatg 1560ggcgcccgca gcctgaccct gaccgtgcag gcccgccagc tgctgagcgg catcgtgcag 1620cagcagaaca acctgctgcg cgccatcgag gcccagcagc acctgctgca gctgaccgtg 1680tggggcatca agcagctgca ggcccgcgtg ctggccgtgg agcgctacct gaaggaccag 1740cagctgctgg gcatctgggg ctgcagcggc aagctgatct gcaccaccgc cgtgccctgg 1800aacgccagct ggagcaacaa gagcctggac cagatctgga acaacatgac ctggatggag 1860tgggagcgcg agatcgacaa ctacaccaac ctgatctaca ccctgatcga ggagagccag 1920aaccagcagg agaagaacga gcaggagctg ctggagctgg acaagtgggc cagcctgtgg 1980aactggttcg acatcagcaa gtggctgtgg tacatcaaga tcttcatcat gatcgtgggc 2040ggcctggtgg gcctgcgcat cgtgttcacc gtgctgagca tcgtgaaccg cgtgcgccag 2100ggctacagcc ccctgagctt ccagacccgc ttccccgccc cccgcggccc cgaccgcccc 2160gagggcatcg aggaggaggg cggcgagcgc gaccgcgacc gcagcagccc cctggtgcac 2220ggcctgctgg ccctgatctg ggacgacctg cgcagcctgt gcctgttcag ctaccaccgc 2280ctgcgcgacc tgatcctgat cgccgcccgc atcgtggagc tgctgggccg ccgcggctgg 2340gaggccctga agtactgggg caacctgctg cagtactgga tccaggagct gaagaacagc 2400gccgtgagcc tgttcgacgc catcgccatc gccgtggccg agggcaccga ccgcatcatc 2460gaggtggccc agcgcatcgg ccgcgccttc ctgcacatcc cccgccgcat ccgccagggc 2520ttcgagcgcg ccctgctgta actcgag 2547 <210> SEQ ID NO 20 <211> LENGTH: 2466<212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223>OTHER INFORMATION: Description of Artificial Sequence:gp160.modSF162.delV2 <400> SEQUENCE: 20gaattcgcca ccatggatgc aatgaagaga gggctctgct gtgtgctgct gctgtgtgga 60gcagtcttcg tttcgcccag cgccgtggag aagctgtggg tgaccgtgta ctacggcgtg 120cccgtgtgga aggaggccac caccaccctg ttctgcgcca gcgacgccaa ggcctacgac 180accgaggtgc acaacgtgtg ggccacccac gcctgcgtgc ccaccgaccc caacccccag 240gagatcgtgc tggagaacgt gaccgagaac ttcaacatgt ggaagaacaa catggtggag 300cagatgcacg aggacatcat cagcctgtgg gaccagagcc tgaagccctg cgtgaagctg 360acccccctgt gcgtgaccct gcactgcacc aacctgaaga acgccaccaa caccaagagc 420agcaactgga aggagatgga ccgcggcgag atcaagaact gcagcttcaa ggtgggcgcc 480ggcaagctga tcaactgcaa caccagcgtg atcacccagg cctgccccaa ggtgagcttc 540gagcccatcc ccatccacta ctgcgccccc gccggcttcg ccatcctgaa gtgcaacgac 600aagaagttca acggcagcgg cccctgcacc aacgtgagca ccgtgcagtg cacccacggc 660atccgccccg tggtgagcac ccagctgctg ctgaacggca gcctggccga ggagggcgtg 720gtgatccgca gcgagaactt caccgacaac gccaagacca tcatcgtgca gctgaaggag 780agcgtggaga tcaactgcac ccgccccaac aacaacaccc gcaagagcat caccatcggc 840cccggccgcg ccttctacgc caccggcgac atcatcggcg acatccgcca ggcccactgc 900aacatcagcg gcgagaagtg gaacaacacc ctgaagcaga tcgtgaccaa gctgcaggcc 960cagttcggca acaagaccat cgtgttcaag cagagcagcg gcggcgaccc cgagatcgtg 1020atgcacagct tcaactgcgg cggcgagttc ttctactgca acagcaccca gctgttcaac 1080agcacctgga acaacaccat cggccccaac aacaccaacg gcaccatcac cctgccctgc 1140cgcatcaagc agatcatcaa ccgctggcag gaggtgggca aggccatgta cgcccccccc 1200atccgcggcc agatccgctg cagcagcaac atcaccggcc tgctgctgac ccgcgacggc 1260ggcaaggaga tcagcaacac caccgagatc ttccgccccg gcggcggcga catgcgcgac 1320aactggcgca gcgagctgta caagtacaag gtggtgaaga tcgagcccct gggcgtggcc 1380cccaccaagg ccaagcgccg cgtggtgcag cgcgagaagc gcgccgtgac cctgggcgcc 1440atgttcctgg gcttcctggg cgccgccggc agcaccatgg gcgcccgcag cctgaccctg 1500accgtgcagg cccgccagct gctgagcggc atcgtgcagc agcagaacaa cctgctgcgc 1560gccatcgagg cccagcagca cctgctgcag ctgaccgtgt ggggcatcaa gcagctgcag 1620gcccgcgtgc tggccgtgga gcgctacctg aaggaccagc agctgctggg catctggggc 1680tgcagcggca agctgatctg caccaccgcc gtgccctgga acgccagctg gagcaacaag 1740agcctggacc agatctggaa caacatgacc tggatggagt gggagcgcga gatcgacaac 1800tacaccaacc tgatctacac cctgatcgag gagagccaga acagcagga gaagaacgag 1860caggagctgc tggagctgga caagtgggcc agcctgtgga actggttcga catcagcaag 1920tggctgtggt acatcaagat cttcatcatg atcgtgggcg gcctggtggg cctgcgcatc 1980gtgttcaccg tgctgagcat cgtgaaccgc gtgcgccagg gctacagccc cctgagcttc 2040cagacccgct tccccgcccc ccgcggcccc gaccgccccg agggctacga ggaggagggc 2100ggcgagcgcg accgcgaccg cagcagcccc ctggtgcacg gcctgctggc cctgatctgg 2160gacgacctgc gcagcctgtg cctgttcagc taccaccgcc tgcgcgacct gatcctgatc 2220gccgcccgca tcgtggagct gctgggccgc cgcggctggg aggccctgaa gtactggggc 2280aacctgctgc agtactggat ccaggagctg aagaacagcg ccgtgagcct gttcgacgcc 2340atcgccatcg ccgtggccga gggcaccgac cgcatcatcg aggtggccca gcgcatcggc 2400cgcgccttcc tgcacatccc ccgccgcatc cgccagggct tcgagcgcgc cctgctgtaa 2460ctcgag 2466 <210> SEQ ID NO 21 <211> LENGTH: 2358 <212> TYPE: DNA <213>ORGANISM: Artificial Sequence <220> FEATURE: <223>OTHER INFORMATION: Description of Artificial Sequence:gp160.modSF162.delV1/V2 <400> SEQUENCE: 21gaattcgcca ccatggatgc aatgaagaga gggctctgct gtgtgctgct gctgtgtgga 60gcagtcttcg tttcgcccag cgccgtggag aagctgtggg tgaccgtgta ctacggcgtg 120cccgtgtgga aggaggccac caccaccctg ttctgcgcca gcgacgccaa ggcctacgac 180accgaggtgc acaacgtgtg ggccacccac gcctgcgtgc ccaccgaccc caacccccag 240gagatcgtgc tggagaacgt gaccgagaac ttcaacatgt ggaagaacaa catggtggag 300cagatgcacg aggacatcat cagcctgtgg gaccagagcc tgaagccctg cgtgaagctg 360acccccctgt gcgtgggcgc cggcaactgc cagaccagcg tgatcaccca ggcctgcccc 420aaggtgagct tcgagcccat ccccatccac tactgcgccc ccgccggctt cgccatcctg 480aagtgcaacg acaagaagtt caacggcagc ggcccctgca ccaacgtgag caccgtgcag 540tgcacccacg gcatccgccc cgtggtgagc acccagctgc tgctgaacgg cagcctggcc 600gaggagggcg tggtgatccg cagcgagaac ttcaccgaca acgccaagac catcatcgtg 660cagctgaagg agagcgtgga gatcaactgc acccgcccca acaacaacac ccgcaagagc 720atcaccatcg gccccggccg cgccttctac gccaccggcg acatcatcgg cgacatccgc 780caggcccact gcaacatcag cggcgagaag tggaacaaca ccctgaagca gatcgtgacc 840aagctgcagg cccagttcgg caacaagacc atcgtgttca agcagagcag cggcggcgac 900cccgagatcg tgatgcacag cttcaactgc ggcggcgagt tcttctactg caacagcacc 960cagctgttca acagcacctg gaacaacacc atcggcccca acaacaccaa cggcaccatc 1020accctgccct gccgcatcaa gcagatcatc aaccgctggc aggaggtggg caaggccatg 1080tacgcccccc ccatccgcgg ccagatccgc tgcagcagca acatcaccgg cctgctgctg 1140acccgcgacg gcggcaagga gatcagcaac accaccgaga tcttccgccc cggcggcggc 1200gacatgcgcg acaactggcg cagcgagctg tacaagtaca aggtggtgaa gatcgagccc 1260ctgggcgtgg cccccaccaa ggccaagcgc cgcgtggtgc agcgcgagaa gcgcgccgtg 1320accctgggcg ccatgttcct gggcttcctg ggcgccgccg gcagcaccat gggcgcccgc 1380agcctgaccc tgaccgtgca ggcccgccag ctgctgagcg gcatcgtgca gcagcagaac 1440aacctgctgc gcgccatcga ggcccagcag cacctgctgc agctgaccgt gtggggcatc 1500aagcagctgc aggcccgcgt gctggccgtg gagcgctacc tgaaggacca gcagctctg 1560ggcatctggg gctgcagcgg caagctgatc tgcaccaccg ccgtgccctg gaacgccagc 1620tggagcaaca agagcctgga ccagatctgg aacaacatga cctggatgga gtgggagcgc 1680gagatcgaca actacaccaa cctgatctac accctgatcg aggagagcca gaaccagcag 1740gagaagaacg agcaggagct gctggagctg gacaagtggg ccagcctgtg gaactggttc 1800gacatcagca agtggctgtg gtacatcaag atcttcatca tgatcgtggg cggcctggtg 1860ggcctgcgca tcgtgttcac cgtgctgagc atcgtgaacc gcgtgcgcca gggctacagc 1920cccctgagct tccagacccg cttccccgcc ccccgcggcc ccgaccgccc cgagggcatc 1980gaggaggagg gcggcgagcg cgaccgcgac cgcagcagcc ccctggtgca cggcctgctg 2040gccctgatct gggacgacct gcgcagcctg tgcctgttca gctaccaccg cctgcgcgac 2100ctgatcctga tcgccgcccg catcgtggag ctgctgggcc gccgcggctg ggaggccctg 2160aagtactggg gcaacctgct gcagtactgg atccaggagc tgaagaacag cgccgtgagc 2220ctgttcgacg ccatcgccat cgccgtggcc gagggcaccg accgcatcat cgaggtggcc 2280cagcgcatcg gccgcgcctt cctgcacatc ccccgccgca tccgccaggg cttcgagcgc 2340gccctgctgt aactcgag 2358 <210> SEQ ID NO 22 <211> LENGTH: 681 <212>TYPE: PRT <213> ORGANISM: Artificial Sequence <220> FEATURE: <223>OTHER INFORMATION: Description of Artificial Sequence:Clade C TV1c8.2 TPA <400> SEQUENCE: 22Met Asp Ala Met Lys Arg Gly Leu Cys Cys Val Leu Leu Leu Cys Gly1               5                   10                  15Ala Val Phe Val Ser Pro Asn Thr Glu Asp Leu Trp Val Thr Val Tyr            20                  25                  30Tyr Gly Val Pro Val Trp Arg Asp Ala Lys Thr Thr Leu Phe Cys Ala        35                  40                  45Ser Asp Ala Lys Ala Tyr Glu Thr Glu Val His Asn Val Trp Ala Thr    50                  55                  60His Ala Cys Val Pro Thr Asp Pro Asn Pro Gln Glu Ile Val Leu Gly65                  70                  75                  80Asn Val Thr Glu Asn Phe Asn Met Trp Lys Asn Asp Met Ala Asp Gln                85                  90                  95Met His Glu Asp Val Ile Ser Leu Trp Asp Gln Ser Leu Lys Pro Cys            100                 105                 110Val Lys Leu Thr Pro Leu Cys Val Thr Leu Asn Cys Thr Asp Thr Asn        115                 120                 125Val Thr Gly Asn Arg Thr Val Thr Gly Asn Ser Thr Asn Asn Thr Asn    130                 135                 140Gly Thr Gly Ile Tyr Asn Ile Glu Glu Met Lys Asn Cys Ser Phe Asn145                 150                 155                 160Ala Thr Thr Glu Leu Arg Asp Lys Lys His Lys Glu Tyr Ala Leu Phe                165                 170                 175Tyr Arg Leu Asp Ile Val Pro Leu Asn Glu Asn Ser Asp Asn Phe Thr            180                 185                 190Tyr Arg Leu Ile Asn Cys Asn Thr Ser Thr Ile Thr Gln Ala Cys Pro        195                 200                 205Lys Val Ser Phe Asp Pro Ile Pro Ile His Tyr Cys Ala Pro Ala Gly    210                 215                 220Tyr Ala Ile Leu Lys Cys Asn Asn Lys Thr Phe Asn Gly Thr Gly Pro225                 230                 235                 240Cys Tyr Asn Val Ser Thr Val Gln Cys Thr His Gly Ile Lys Pro Val                245                 250                  255Val Ser Thr Gln Leu Leu Leu Asn Gly Ser Leu Ala Glu Glu Gly Ile            260                 265                 270Ile Ile Arg Ser Glu Asn Leu Thr Glu Asn Thr Lys Thr Ile Ile Val        275                 280                 285His Leu Asn Glu Ser Val Glu Ile Asn Cys Thr Arg Pro Asn Asn Asn    290                 295                 300Thr Arg Lys Ser Val Arg Ile Gly Pro Gly Gln Ala Phe Tyr Ala Thr305                 310                 315                 320Asn Asp Val Ile Gly Asn Ile Arg Gln Ala His Cys Asn Ile Ser Thr                325                 330                 335Asp Arg Trp Asn Lys Thr Leu Gln Gln Val Met Lys Lys Leu Gly Glu            340                 345                 350His Phe Pro Asn Lys Thr Ile Gln Phe Lys Pro His Ala Gly Gly Asp        355                 360                 365Leu Glu Ile Thr Met His Ser Phe Asn Cys Arg Gly Glu Phe Phe Tyr    370                 375                 380Cys Asn Thr Ser Asn Leu Phe Asn Ser Thr Tyr His Ser Asn Asn Gly385                 390                 395                 400Thr Tyr Lys Tyr Asn Gly Asn Ser Ser Ser Pro Ile Thr Leu Gln Cys                405                 410                 415Lys Ile Lys Gln Ile Val Arg Met Trp Gln Gly Val Gly Gln Ala Thr            420                 425                 430Tyr Ala Pro Pro Ile Ala Gly Asn Ile Thr Cys Arg Ser Asn Ile Thr        435                 440                 445Gly Ile Leu Leu Thr Arg Asp Gly Gly Phe Asn Thr Thr Asn Asn Thr    450                 455                 460Glu Thr Phe Arg Pro Gly Gly Gly Asp Met Arg Asp Asn Trp Arg Ser465                 470                 475                 480Glu Leu Tyr Lys Tyr Lys Val Val Glu Ile Lys Pro Leu Gly Ile Ala                485                 490                 495Pro Thr Lys Ala Ile Ser Ser Val Val Gln Ser Glu Lys Ser Ala Val            500                 505                 510Gly Ile Gly Ala Val Phe Leu Gly Phe Leu Gly Ala Ala Gly Ser Thr        515                 520                 525Met Gly Ala Ala Ser Ile Thr Leu Thr Val Gln Ala Arg Gln Leu Leu    530                 535                 540Ser Gly Ile Val Gln Gln Gln Ser Asn Leu Leu Lys Ala Ile Glu Ala545                 550                 555                 560Gln Gln His Met Leu Gln Leu Thr Val Trp Gly Ile Lys Gln Leu Gln                565                 570                 575Ala Arg Val Leu Ala Ile Glu Arg Tyr Leu Lys Asp Gln Gln Leu Leu            580                 585                 590Gly Ile Trp Gly Cys Ser Gly Arg Leu Ile Cys Thr Thr Ala Val Pro        595                 600                 605Trp Asn Ser Ser Trp Ser Asn Lys Ser Glu Lys Asp Ile Trp Asp Asn    610                 615                 620Met Thr Trp Met Gln Trp Asp Arg Glu Ile Ser Asn Tyr Thr Gly Leu625                 630                 635                 640Ile Tyr Asn Leu Leu Glu Asp Ser Gln Asn Gln Gln Glu Lys Asn Glu                645                 650                 655Lys Asp Leu Leu Glu Leu Asp Lys Trp Asn Asn Leu Trp Asn Trp Phe            660                 665                 670Asp Ile Ser Asn Trp Pro Trp Tyr Ile         675                 680<210> SEQ ID NO 23 <211> LENGTH: 681 <212> TYPE: PRT <213>ORGANISM: Artificial Sequence <220> FEATURE: <223>OTHER INFORMATION: Description of Artificial Sequence: Clade B SF162 TPA<400> SEQUENCE: 23Met Asp Ala Met Lys Arg Gly Leu Cys Cys Val Leu Leu Leu Cys Gly1               5                   10                  15Ala Val Phe Val Ser Pro Ser Ala Val Glu Lys Leu Trp Val Thr Val            20                  25                  30Tyr Tyr Gly Val Pro Val Trp Lys Glu Ala Thr Thr Thr Leu Phe Cys        35                  40                  45Ala Ser Asp Ala Lys Ala Tyr Asp Thr Glu Val His Asn Val Trp Ala    50                  55                  60Thr His Ala Cys Val Pro Thr Asp Pro Asn Pro Gln Glu Ile Val Leu65                  70                  75                  80Glu Asn Val Thr Glu Asn Phe Asn Met Trp Lys Asn Asn Met Val Glu                85                  90                  95Gln Met His Glu Asp Ile Ile Ser Leu Trp Asp Gln Ser Leu Lys Pro            100                 105                 110Cys Val Lys Leu Thr Pro Leu Cys Val Thr Leu His Cys Thr Asn Leu        115                 120                 125Lys Asn Ala Thr Asn Thr Lys Ser Ser Asn Trp Lys Glu Met Asp Arg    130                 135                 140Gly Glu Ile Lys Asn Cys Ser Phe Lys Val Thr Thr Ser Ile Arg Asn145                 150                 155                 160Lys Met Gln Lys Glu Tyr Ala Leu Phe Tyr Lys Leu Asp Val Val Pro                165                 170                 175Ile Asp Asn Asp Asn Thr Ser Tyr Lys Leu Ile Asn Cys Asn Thr Ser            180                 185                 190Val Ile Thr Gln Ala Cys Pro Lys Val Ser Phe Glu Pro Ile Pro Ile        195                 200                 205His Tyr Cys Ala Pro Ala Gly Phe Ala Ile Leu Lys Cys Asn Asp Lys    210                 215                 220Lys Phe Asn Gly Ser Gly Pro Cys Thr Asn Val Ser Thr Val Gln Cys225                 230                 235                 240Thr His Gly Ile Arg Pro Val Val Ser Thr Gln Leu Leu Leu Asn Gly                245                 250                 255Ser Leu Ala Glu Glu Gly Val Val Ile Arg Ser Glu Asn Phe Thr Asp            260                 265                 270Asn Ala Lys Thr Ile Ile Val Gln Leu Lys Glu Ser Val Glu Ile Asn        275                 280                 285Cys Thr Arg Pro Asn Asn Asn Thr Arg Lys Ser Ile Thr Ile Gly Pro    290                 295                 300Gly Arg Ala Phe Tyr Ala Thr Gly Asp Ile Ile Gly Asp Ile Arg Gln305                 310                 315                 320Ala His Cys Asn Ile Ser Gly Glu Lys Trp Asn Asn Thr Leu Lys Gln                325                 330                 335Ile Val Thr Lys Leu Gln Ala Gln Phe Gly Asn Lys Thr Ile Val Phe            340                 345                 350Lys Gln Ser Ser Gly Gly Asp Pro Glu Ile Val Met His Ser Phe Asn        355                 360                 365Cys Gly Gly Glu Phe Phe Tyr Cys Asn Ser Thr Gln Leu Phe Asn Ser    370                 375                 380Thr Trp Asn Asn Thr Ile Gly Pro Asn Asn Thr Asn Gly Thr Ile Thr385                 390                 395                 400Leu Pro Cys Arg Ile Lys Gln Ile Ile Asn Arg Trp Gln Glu Val Gly                405                 410                 415Lys Ala Met Tyr Ala Pro Pro Ile Arg Gly Gln Ile Arg Cys Ser Ser            420                 425                 430Asn Ile Thr Gly Leu Leu Leu Thr Arg Asp Gly Gly Lys Glu Ile Ser        435                 440                 445Asn Thr Thr Glu Ile Phe Arg Pro Gly Gly Gly Asp Met Arg Asp Asn    450                 455                 460Trp Arg Ser Glu Leu Tyr Lys Tyr Lys Val Val Lys Ile Glu Pro Leu465                 470                 475                 480Gly Val Ala Pro Thr Lys Ala Ile Ser Ser Val Val Gln Ser Glu Lys                485                 490                 495Ser Ala Val Thr Leu Gly Ala Met Phe Leu Gly Phe Leu Gly Ala Ala            500                 505                 510Gly Ser Thr Met Gly Ala Arg Ser Leu Thr Leu Thr Val Gln Ala Arg        515                 520                 525Gln Leu Leu Ser Gly Ile Val Gln Gln Gln Asn Asn Leu Leu Arg Ala    530                 535                 540Ile Glu Ala Gln Gln His Leu Leu Gln Leu Thr Val Trp Gly Ile Lys545                 550                 555                 560Gln Leu Gln Ala Arg Val Leu Ala Val Glu Arg Tyr Leu Lys Asp Gln                565                 570                 575Gln Leu Leu Gly Ile Trp Gly Cys Ser Gly Lys Leu Ile Cys Thr Thr            580                 585                 590Ala Val Pro Trp Asn Ala Ser Trp Ser Asn Lys Ser Leu Asp Gln Ile        595                 600                 605Trp Asn Asn Met Thr Trp Met Glu Trp Glu Arg Glu Ile Asp Asn Tyr    610                 615                 620Thr Asn Leu Ile Tyr Thr Leu Ile Glu Glu Ser Gln Asn Gln Gln Glu625                 630                 635                 640Lys Asn Glu Gln Glu Leu Leu Glu Leu Asp Lys Trp Ala Ser Leu Trp                645                 650                 655Asn Trp Phe Asp Ile Ser Lys Trp Leu Trp Tyr Ile            660                 665

1-70. (canceled)
 71. An injectable immunogenic composition comprising anEnv polypeptide complexed to a polyanionic carbomer polymer.
 72. Animmunogenic composition comprising an Env polypeptide complexed to apolyanionic carbomer polymer, wherein the concentration of thepolyanionic carbomer polymer is between about 0.01% (w/v) and about 0.5%(w/v).
 73. The immunogenic composition of claim 71, wherein theconcentration of the polyanionic carbomer polymer is between about 0.01%(w/v) and about 0.5% (w/v).
 74. The immunogenic composition of claim 71,wherein the polyanionic polymer comprises CARBOPOL 971P NF™, CARBOPOL974P NF™, or combinations thereof.
 75. The immunogenic composition ofclaim 72, wherein the polyanionic polymer comprises CARBOPOL 971P NF™,CARBOPOL 974P NF™, or combinations thereof.
 76. The immunogeniccomposition of claim 71, wherein the Env polypeptide comprises apolypeptide selected from the group consisting of a gp160 Envpolypeptide, a polypeptide derived from a gp160 Env polypeptide, a gp140Env polypeptide, a polypeptide derived from a gp140 Env polypeptide, agp120 Env polypeptide, and a polypeptide derived from a gp120 Envpolypeptide.
 77. The immunogenic composition of claim 71, wherein theEnv polypeptide is an HIV Env polypeptide and the composition furthercomprises a second Env polypeptide selected from a different HIV subtypeas the Env polypeptide wherein the Env polypeptide and the second Envpolypeptide are derived from an HIV subtype B strain and an HIV subtypeC strain or vice-versa.
 78. The immunogenic composition of claim 71further comprising an adjuvant which is an oil-in-water emulsion.
 79. Amethod of generating an immunogenic composition comprising an Envpolypeptide complexed to a polyanionic carbomer polymer, the methodcomprising: (a) contacting the polyanionic carbomer polymer with the Envpolypeptide under conditions where the pH is below the pI of the Envpolypeptide in a solution; (b) incubating the polyanionic carbomerpolymer with the Env polypeptide together to allow the Env polypeptideto form a complex with the polyanionic carbomer polymer.
 80. The methodof claim 79, wherein the pH is between 3 and
 5. 81. The method of claim79, wherein the pH is between 3 and
 4. 82. The method of claim 79,wherein the concentration of the polyanionic carbomer polymer aftercontacting step (a) is between about 0.01% (w/v) and about 0.5% (w/v).83. The method of claim 79, wherein the polyanionic carbomer polymercomprises CARBOPOL 971P NF™, CARBOPOL 974P NF™, or combinations thereof.84. The method of claim 79, wherein the Env polypeptide comprises apolypeptide selected from the group consisting of a gp160 Envpolypeptide, a polypeptide derived from a gp160 Env polypeptide, a gp140Env polypeptide, a polypeptide derived from a gp140 Env polypeptide, agp120 Env polypeptide, and a polypeptide derived from a gp120 Envpolypeptide.
 85. The method of claim 79, wherein the Env polypeptide isan HIV Env polypeptide and the composition further comprises a secondHIV Env polypeptide selected from a different HIV subtype as the Envpolypeptide wherein the Env polypeptide and the second Env polypeptideare derived from an HIV subtype B strain and an HIV subtype C strain orvice-versa.
 86. The method of claim 79, further comprising adding anadjuvant, which is an oil-in-water emulsion adjuvant, to the solution.87. A method of generating an immune response in a subject, comprisingadministering to said subject an immunogenic composition comprising anEnv polypeptide complexed to a polyanionic carbomer polymer, therebygenerating the immune response to the Env polypeptide.
 88. The method ofclaim 87, wherein the immunogenic composition is administeredintramuscularly, intramucosally, intranasally, subcutaneously,intradermally, transdermally, orally or intravenously.
 89. The method ofclaim 87, wherein the immunogenic composition is administered byinjection.
 90. The method of claim 87, wherein the concentration of thepolyanionic carbomer polymer is between about 0.01% (w/v) and about 0.5%(w/v).